U.S. patent application number 10/239656 was filed with the patent office on 2004-02-26 for multifunctional polypeptides comprising a binding site to an epitope of the nkg2d receptor complex.
Invention is credited to Borschert, Katrin, Hofmeister, Robert, Kischel, Roman, Kufer, Peter, Lutterbuse, Ralf, Mayer, Monika, Rietmuller, Gert.
Application Number | 20040038339 10/239656 |
Document ID | / |
Family ID | 8168233 |
Filed Date | 2004-02-26 |
United States Patent
Application |
20040038339 |
Kind Code |
A1 |
Kufer, Peter ; et
al. |
February 26, 2004 |
Multifunctional polypeptides comprising a binding site to an
epitope of the nkg2d receptor complex
Abstract
The present invention relates to a multifunctional polypeptide
comprising a first domain comprising a binding site specifically
recognizing an extracellular epitope of the NKG2D receptor complex
and a second domain having receptor or ligand function.
Furthermore, the present invention relates to polynucleotides
encoding the multifunctional polypeptide, to vectors comprising
said polypeptides and to cells comprising said polypeptides or said
vectors. The invention also relates to compositions comprising
either of the above recited molecules, alone or in combination, as
well as to specific medical uses of the multifunctional polypeptide
of the invention.
Inventors: |
Kufer, Peter; (Moosburg,
DE) ; Rietmuller, Gert; (Munich, DE) ;
Lutterbuse, Ralf; (Munich, DE) ; Borschert,
Katrin; (Munich, DE) ; Kischel, Roman;
(Munich, DE) ; Mayer, Monika; (Ottobeuren, DE)
; Hofmeister, Robert; (Germering, DE) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Family ID: |
8168233 |
Appl. No.: |
10/239656 |
Filed: |
January 7, 2003 |
PCT Filed: |
March 26, 2001 |
PCT NO: |
PCT/EP01/03414 |
Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.5 |
Current CPC
Class: |
A61P 1/16 20180101; A61P
27/02 20180101; A61P 37/06 20180101; A61P 31/10 20180101; A61P
33/10 20180101; A61P 37/00 20180101; C07K 2317/31 20130101; C07K
2319/00 20130101; A61P 37/02 20180101; C07K 2317/34 20130101; A61K
2039/53 20130101; A61P 35/02 20180101; A61P 37/08 20180101; A61P
13/12 20180101; A61P 31/04 20180101; A61P 3/10 20180101; A61P 13/08
20180101; A61P 5/14 20180101; A61P 31/00 20180101; A61P 33/02
20180101; A61P 21/04 20180101; A61P 25/00 20180101; A61P 29/00
20180101; A61P 35/00 20180101; C07K 2317/622 20130101; A61P 17/00
20180101; C07K 16/2851 20130101; A61P 31/12 20180101; A61P 19/02
20180101 |
Class at
Publication: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.5 |
International
Class: |
C12P 021/02; C12N
005/06; C07K 014/705; C07H 021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 24, 2000 |
EP |
00106467.4 |
Claims
1. A multifunctional polypeptide comprising (a) a first domain
comprising a binding site specifically recognizing an extracellular
epitope of the NKG2D receptor complex; and (b) a second domain
having receptor or ligand function.
2. The multifunctional polypeptide of claim 1 wherein said binding
site is the binding site of an immunoglobulin chain.
3. The multifunctional polypeptide of claim 1 wherein said binding
site is a natural NKG2D-ligand of said receptor complex.
4. The multifunctional polypeptide of claim 3 wherein said natural
NKG2ligand is selected from the group consisting of MIC-A, MIC-B,
ULBP-1 and ULBP-2.
5. The multifunctional polypeptide of claims 1 to 4 wherein said
binding site specifically recognizes an extracellular epitope of
NKG2D or of DAP10.
6. The multifunctional polypeptide of any one of claims 1 to 5
wherein said receptor or ligand function is an antigen binding site
of antibodies or fragments or derivatives thereof against (i) tumor
associated antigens, (ii) antigens of infective agents or (iii)
surface markers of sub-populations of cells such as differentiation
antigens (CD antigens), natural ligands or receptors or fragments
thereof or modifications thereof that interact with said tumor
associated antigens or surface markers, preferably (i) heregulins,
binding to the tumor associated antigens erbB-2,-3 and -4, (ii) CD4
that interacts with gp 120 of HIV infected cells or (iii)
melanocyte stimulating hormone (MSH) that binds to the MSH receptor
on melanocytes and tumors derived therefrom (maligned melanomes) or
chemokines binding to corresponding chemokine receptors, or MHC
molecules or fragments thereof complexed with peptides that bind to
T-cell receptors of predefined specificity and thus recognize
certain T-cell sub-populations or antigen binding sites of T-cell
receptors that specifically interact with predefined MHC peptide
complexes or NKp46 which interacts with haemagglutinin (HA) of
influenza virus.
7. The multifunctional polypeptide of claim 6 wherein said
tumor-associated antigen is selected from the group consisting of
Lewis Y, Muc-1, erbB-2,-3 and -4, Ep-CAM, EGF-receptor (e.g. EGFR
type I or EGFR type II), EGFR deletion neoepitope, CA19-9, Muc-1,
LeY, TF-, Tn- and sTn-antigen, TAG-72, PSMA, STEAP, Cora antigen,
CD7, CD19 and CD20, CD22, CD25, Ig-.alpha. and Ig-.beta., A33 and
G250, CD30, MCSP and gp100, CD44-v6, MT-MMPs, (MIS) receptor type
II, carboanhydrase 9, F19-antigen, Ly6, desmoglein 4, PSCA, Wue-1,
GD2 and GD3 as well as TM4SF-antigens (CD63, L6, CO-29, SAS) or the
alpha and gamma subunit of the fetal type acetylcholinreceptor
(AChR).
8. The multifunctional polypeptide of claim 6 wherein said surface
marker for an infected cell is selected from the group consisting
of viral envelope antigens, e.g. of human retroviruses (HTLV I and
II, HIV1 and 2) or human herpes viruses (HSV1 and 2, CMV, EBV),
haemagglutinin e.g. of influenza virus A, B or C, glycoprotein E1
and E2 of rubella virus or RGP of rabies virus.
9. The multifunctional polypeptide of any one of claims 1 to 8
which is a bi-specific antibody.
10. The multifunctional polypeptide of claim 9 wherein said
multifunctional polypeptide is selected from the group consisting
of a synthetic, a chimeric and a humanized antibody.
11. The multifunctional polypeptide of any one of claims 1 to 10
which is a single-chain.
12. The multifunctional polypeptide of any one of claims 1 to 10
wherein said two domains are connected by a polypeptide linker.
13. The multifunctional polypeptide of any one of claims 1 to 12
wherein said first and/or second domain mimic or correspond to a
V.sub.H and V.sub.L region of a natural antibody.
14. The multifunctional polypeptide of any one of claims 1 to 13
wherein at least one of said domains is a single-chain fragment of
the variable region of the antibody.
15. The multifunctional polypeptide of any one of claims 1 to 14
wherein said domains are ranged in the order
V.sub.LNKG2D-V.sub.HNKG2D-V.sub.HTA-- V.sub.L-TA or
V.sub.LTA-V.sub.HTA-V.sub.HNKG2D-V.sub.LNKG2D, wherein the TA
represents a target antigen.
16. The multifunctional polypeptide of claim 15 wherein said target
antigen is selected from the group consisting of Lewis Y, Muc-1,
erbB-2,-3 and -4, Ep-CAM, EGF-receptor (e.g. EGFR type I or EGFR
type II), EGFR deletion neoepitope, CA19-9, Muc-1, LeY, TF-, Tn- -
and sTn-antigen, TAG-72, PSMA, STEAP, Cora antigen, CD7, CD19 and
CD20, CD22, CD25, Ig-.alpha. and Ig-.beta., A33 and G250, CD30,
MCSP and gp100, CD44-v6, MT-MMPs, (MIS) receptor type II,
carboanhydrase 9, F19-antigen, Ly6, desmoglein 4, PSCA, Wue-1, GD2
and GD3 as well as TM4SF-antigens (CD63, L6, CO-29, SAS) or the
alpha and gamma subunit of the fetal type acetylcholinreceptor
(AChR).
17. The multifunctional polypeptide of any one of claims 12 to 16
wherein said polypeptide linker comprises a plurality of glycine,
serine and/or alanine residues.
18. The multifunctional polypeptide of any one of claims 12 to 17
wherein said polypeptide linker comprises a plurality of
consecutive copies of an amino acid sequence.
19. The polypeptide of any one of claims 12 to 18 wherein said
polypeptide linker comprises 1 to 5, 5 to 10 or 10 to 15 amino acid
residues.
20. The multifunctional polypeptide of any one of claims 4 to 19
wherein said polypeptide linker comprises the amino acid sequence
Gly-Gly-Gly-Gly-Ser.
21. The multifunctional polypeptide of any one of claims 1 to 20
comprising at least one further domain.
22. The multifunctional polypeptide of claim 21 wherein said
further domain is linked by covalent or non-covalent bonds.
23. The multifunctional polypeptide of claims 21 or 22, wherein
said at least one further domain comprises an effector molecule
having a conformation suitable for biological activity, capable of
sequestering an ion or selective binding to a solid support or to a
preselected determinant.
24. The multifunctional polypeptide of any one of claims 21 to 23
wherein said further domain confers a co-stimulatory and/or a
co-activating function.
25. The multifunctional polypeptide of claim 24 wherein said
co-stimulatory function is mediated by a CD28-ligand or a
CD137-ligand.
26. The multifunctional polypeptide of claim 25 wherein said
CD28-ligand or CD137-ligand is B7-1 (CD80), B7-2 (CD86), an aptamer
or an antibody or a functional fragment or a functional derivative
thereof.
27. A polynucleotide which upon expression encodes a
multifunctional polypeptide and/or functional parts of a
multifunctional polypeptide of any one of claims 1 to 26.
28. A vector comprising the polynucleotide of claim 27.
29. A cell transfected with the polynucleotide of claim 27 or the
vector of claim 28.
30. A method for the preparation of the multifunctional polypeptide
and/or parts of the multifunctional polypeptide of any one of
claims 1 to 26 comprising culturing a cell of claim 29 and
isolating said multifunctional polypeptide or functional parts
thereof from the culture.
31. A composition comprising the polypeptide of any one of claims 1
to 26, the polynucleotide of claim 27 or the vector of claim
28.
32. The composition of claim 31 further comprising a molecule
conferring a co-stimulatory and/or co-activating function.
33. The composition of claim 31 wherein said co-stimulatory
function is mediated by a CD28-ligand or a CD137-ligand.
34. The composition of claim 31 wherein said CD28-ligand or
CD137-ligand is B7-1 (CD80), B7-2 (CD86), an aptamer or an antibody
or a functional fragment or a functional derivative thereof.
35. The composition of any one of claims 31 to 34 which is a
pharmaceutical composition optionally further comprising a
pharmaceutically acceptable carrier.
36. The composition of any one of claims 31 to 35 which is a
diagnostic composition optionally further comprising suitable means
for detections.
37. Use of the multifunctional polypeptide of any one of claims 1
to 26, the polynucleotide of claim 27 or the vector of claim 28 for
the preparation of a pharmaceutical composition for the treatment
of cancer, infections and/or autoimmune conditions, cancer, i.e.
maligne (solid) tumors and hematopoietic cancer forms (leukemias
and lymphomas), benigne tumors such as benigne hyperplasia of the
prostate gland (BPH), autonomous adenomes of the thyroid gland or
of other endocrine glands or adenomas of the colon; initial stages
of the malignancies, infectious diseases, caused by viruses,
bacteria, fungi, protozoa or helmints, auto immune diseases wherein
the elimination of the subpopulation of immune cells is desired
that causes the disease; prevention of transplant rejection or
allergies.
38. The use of claim 37 wherein said infection is a viral, a
bacterial or a fungal infection, wherein said cancer is a head and
neck cancer, gastric cancer, oesaphagus cancer, stomach cancer,
colorectal cancer, coloncarcinomaa, cancer of liver and
intrahepatic bile ducts, pancreatic cancer, lung cancer, small cell
lung cancer, cancer of the larynx, breast cancer, mamma carcinoma,
malignant melanoma, multiple myeloma, sarcomas, rhabdomyosarcoma,
lymphomas, folicular non-Hodgkin-lymphoma, leukemias, T- and
B-cell-leukemias, Hodgkin-lymphoma, B-cell lymphoma, ovarian
cancer, cancer of the uterus, cervical cancer, prostate cancer,
genital cancer, renal cancer, cancer of the testis, thyroid cancer,
bladder cancer, plasmacytoma or brain cancer or wherein said
autoimmune condition is ankylosing spondylitis, acute anterior
uveitis, Goodpasture's syndrome, Multiple sclerosis, Graves'
disease, Myasthenia gravis, Systemic lupus erythematosus,
Insulin-dependent diabetes mellitus, Rheumatoid arthritis,
Pemphigus vulgaris, Hashimoto's thyroiditis or autoimmune
Hepatitis.
39. Use of the polynucleotide of claim 27 or the vector of claim 28
for the preparation of a composition for gene therapy.
40. A method for the treatment of cancer, infections or autoimmune
conditions comprising introducing the polypeptide of any one of
claims 1 to 26, the polynucleotide of claim 27 or the vector of
claim 28 or the composition of claim 35 into a mammal affected by
said malignancies or diseases.
41. A method for delaying a pathological condition comprising
introducing the polypeptide of any one of claims 1 to 26, the
polynucleotide of claim 27 or the vector of claim 28 or the
composition of claim 35 into a mammal affected by said pathological
condition.
42. The method of claim 40 or 41 wherein said mammal is a
human.
43. A kit comprising the multifunctional polypeptide of any one of
claims 1 to 26, the polynucleotide of claim 27, the vector of claim
28, the cell of claim 29 or the composition of any one of claims 31
to 36.
Description
[0001] The present invention relates to a multifunctional
polypeptide comprising a first domain comprising a binding site
specifically recognizing an extracellular epitope of the NKG2D
receptor complex and a second domain having receptor or ligand
function. Furthermore, the present invention relates to
polynucleotides encoding the multifunctional polypeptide, to
vectors comprising said polypeptides and to cells comprising said
polynucleotides or said vectors. The invention also relates to
compositions comprising either of the above recited molecules,
alone or in combination, as well as to specific medical uses of the
multifunctional polypeptide of the invention.
[0002] Several documents are cited throughout the text of this
specification. The disclosure content of each of these documents
(including any manufacturer's specifications, instructions etc.) is
herewith incorporated by reference.
[0003] Many multifunctional polypeptide compounds described in the
prior art are bispecific antibodies of varying molecular formats
developed for retargeting immune effector cells against malignant
or infected target cells, clearing pathogens or autoantibodies from
blood circulation, enhancing drug therapy or as vaccines or as
carriers e.g. of radioisotopes. Bispecific antibodies designed to
redirect the cytotoxic activity of immune effector cells against
target cells usually comprise a binding site recognizing a
tumor-associated or a viral antigen on the target cells and a
second binding site that interacts with a triggering molecule on
the effector cells. Among the effector cells recruited in the prior
art by bispecific antibody approaches were T-lymphocytes, NK-cells,
monocytes and polymorphonuclear neutrophils. Triggering molecules
for bispecific antibodies were usually selected from a group of
cell surface receptors consisting of CD64, CD16, the
.alpha./.beta.-T cell receptor (TCR) and CD3, but also alternative
triggering molecules like CD2, CD89, CD32, CD44, CD69 and the
TCR-zeta chain were evaluated. Bispecific antibodies capable of
redirecting cytotoxic T-lymphocytes (phenotype:
CD3.sup.+/CD56.sup.-CD8.sup.+) to target cells either contain a
binding site for the TCR, CD3, the zeta-chain or CD2. By engaging
one of these triggering molecules, however, antigen specific
signaling via the TCR-complex is disturbed since either epitopes of
the TCR-complex itself are involved (the TCR, CD3 or the
zeta-chain) or in case of CD2 a molecule that directly contributes
to the TCR-signal by coaggregation of the src-related protein
tyrosine kinase lck, associated with its cytoplasmic tail, with the
TCR-complex.
[0004] Thus, the technical problem was to provide multifunctional
polypeptides that enhance the specific activation of lymphocytes in
the direct neighborhood of disease-related cells without
interfering with the receptor specificity and/or function of those
cytolytic lymphocytes.
[0005] The solution to said technical problem is achieved by
providing the embodiments characterized in the claims.
[0006] Accordingly, the present invention relates to a
multifunctional polypeptide comprising a first domain comprising a
binding site specifically recognizing an extracellular epitope of
the NKG2D receptor complex and a second domain having receptor or
ligand function.
[0007] The term "multifunctional polypeptide" in connection with
the present invention means a polypeptide that effects under
suitable (also in vitro) conditions, such as physiological
including pathological, such as in vivo or ex vivo conditions at
least two, such as three, four, five or six different biological
functions. Physiological in vitro conditions include buffered
solutions, such as phosphate buffered solutions in the pH range of
5 to 9 and can be further derived from the appended examples. These
functions are as specified further below. They include binding of
the specified domains with the molecules further specified herein.
Binding may subsequently trigger a further biological function
including the onset of a cascade, binding to receptors, modulation
of signaling pathways or of gene expression and/or influence on
apoptotic cell-death. At least two of these domains conferring
differing biological functions and preferably the two domains
specified herein above do not naturally occur together, i.e. do not
naturally occur in this configuration or at all on the same
polypeptide or protein or protein complex.
[0008] The term "receptor or ligand function" refers to a naturally
occurring or non-naturally occurring binding function of a molecule
such as a naturally occurring receptor that is preferably located
on a cell surface with a fitting ligand; Examples of such
receptor/ligand pairs are antibodies/antigens or other members of
the Ig superfamily and their corresponding ligands or hormone
receptors/hormones or carbohydrate/lectin interactions. Ligands in
general, but not exclusively, refer to molecules that have a
natural binding partner. In correspondence with the above, they may
be antigens or hormones. However, they may also be of non-natural
configuration or origin. Receptors/ligands as described above may
be of natural origin, of recombinant or (semi) synthetic
origin.
[0009] NKG2D is a C-type lectin-like NK cell receptor (Houchins
(1991) J.Exp.Med. 172:1017) that forms the NKG2D receptor complex
together with DAP10 (Wu (1999) Science 285: 730). DAP10 carries an
activating sequence motif for Pl.sub.3-kinase in its cytoplasmic
domain and acts as signal transduction module for NKG2D that lacks
signaling motifs in its cytoplasmic domain. Engagement of this
receptor complex triggers a signaling cascade capable of inducing
NK cell cytotoxicity. Like other NK cell receptors, the NKG2D
receptor complex was also found to be expressed in certain T cell
subsets, namely .gamma./.delta.-T cells, CD8.sup.+ .alpha./.beta.-T
cells and in a diminishing minority of CD4.sup.+ .alpha./.beta.-T
cells (Bauer (1999) Science 285: 727).
[0010] NK cells are dominant effectors of humoral immune responses,
that gain antigen specificity through binding of IgG-antibodies to
their surface Fc.gamma.-receptor CD16. Thus, CD16 acts as specific
antigen receptor enabling antibody-armed NK cells to destroy target
cells in an antigen specific manner. T-lymphocytes are the
effectors of cellular immune responses, that carry the TCR-complex
as specific antigen receptor. The TCR-complex is composed of
several invariant chains including the CD3-complex and the zeta
chain as well as two variable chains that confer the clonotypic
antigen specificity. Depending on the type of variable chains found
in the TCR-complex (either .alpha.- and .beta.-chain or .gamma.-
and .delta.-chain), T-lymphocytes can be divided into
.alpha./.beta.- and .gamma./.delta.-T cells. TCR-mediated
recognition of target cells by cytotoxic T-lymphocytes i.e.;
CD8.sup.+ .alpha./.beta.-T cells and .gamma./.delta.-T cells
usually leads to target cell lysis.
[0011] The majority of known lymphocyte-directed bispecific
antibodies either recruit NK cells or T cells only. NK cells are
usually recruited through engagement of CD16, forming the major
extracellular part of the Fc.gamma.-receptor IIIA complex, while T
cell recruitment is usually mediated through engagement of CD3, an
invariant multi-chain component of the T cell receptor (TCR).
Bispecific antibodies directed at the zeta chain associated with
CD16 on NK cells as well as with the TCR on T cells, are capable of
engaging both types of effector lymphocytes (WO00/03016). However,
bispecific antibodies directed at the zeta chain, like those
directed at CD3, also activate non-cytotoxic CD4.sup.+ T cells,
that in vivo unlike CD8.sup.+ T cells contribute to undesired side
effects e.g. due to systemic cytokine release without essentially
contributing to the cytotoxic elimination of target cells.
[0012] The NKG2D-specific multifunctional molecules of the
invention (which are in preferred embodiments bifunctional
molecules comprising said first and second domain referred to
above) in contrast to lymphocyte-directed bispecific antibodies
known in the prior art are capable of recruiting with exceptional
precision the entire range of lymphocytes that naturally carry a
cytotoxic phenotype i.e. NK cell, CD8.sup.+ .alpha./.beta.-T cells
and .gamma./.delta.-T cells without essentially touching other cell
types like CD4.sup.+ .alpha./.beta.-T cells that are usually not
cytotoxic.
[0013] The term "recruitment of cytotoxic lymphocytes" as used in
the present invention is not limited to redirected lysis but also
comprises enhancement of cytotoxicity and T-cell priming.
[0014] Thus, the NKG2D-directed molecules of the invention are
unique due to their precision of exhaustively but also exclusively
recruiting all relevant cytotoxic lymphocytes. In further contrast
to lymphocyte-directed bispecific antibodies known in the prior
art, the multifunctional molecules of the invention neither
directly nor indirectly engage the specific antigen receptors of
cytotoxic lymphocytes including the upstream cytoplasmic steps of
the corresponding signaling cascades. In other words, function of
the T-cell receptor complex is not impaired since the
multifunctional polypeptide of the invention does not bind thereto.
The signaling cascade downstream of the signal conferred by the
T-cell receptor is therefore not affected by the interaction with
the multifunctional polypeptide of the invention. As a result,
activation and/or proliferation of cytotoxic lymphocytes is
selectively supported, that due to their antigen receptor
specificity are engaged in a specific immune response against those
target cells recognized by the multifunctional molecules of the
invention.
[0015] Said upstream signaling cascade in T- and NK-cells comprises
ITAM polypeptides, Src kinases, ZAP-70/Syk and adaptor proteins
such as LAT and SLP-76 responsible for the recruitment of effector
molecules of the downstream signaling cascade. The downstream
signaling cascade comprises molecules like the Pl3-kinase as well
as PLC.gamma., Grb2, Vav, Cbl and Nck.
[0016] By avoiding the engagement of specific antigen receptors
and/or the upstream cytoplasmic steps of the corresponding
signaling cascades the multifunctional molecules of the invention
advantageously interfere to a smaller degree with specific antigen
recognition than other lymphocyte-directed bispecific antibodies
known in the prior art that e.g. bind to CD16 of the
Fc.gamma.-receptor complex on NK cells or to the CD3-component of
the TCR-complex on T-lymphocytes. In particular, lymphocyte
effector functions mediated by a target cell specific immune
response may be overruled through engagement of specific antigen
receptors and/or the upstream cytoplasmic steps of the
corresponding signaling cascades by bispecific antibodies of the
prior art. In contrast, the multifunctional molecules of the
invention by engaging the NKG2D receptor complex, which is neither
directly associated with specific antigen receptors nor with the
upstream steps of their cytoplasmic signaling cascades, are capable
of enhancing the activation of those cytotoxic lymphocytes that
recognize the same target cell through their specific antigen
receptor.
[0017] This explains the surprising result described in the
appended examples, that an NKG2D-mediated signal can accelerate
priming of naive CD8.sup.+ T-cells even in the presence of
[0018] (i) a strong primary signal mediated through engagement of
the antigen specific T-cell receptor complex and
[0019] (ii) maximum co-stimulation provided by B7-1, the dominant
mediator of the second T-cell signal.
[0020] Furthermore, it was surprisingly found, that the
cytotoxicity of CD8.sup.+ T-cells and NK-cells triggered by the
engagement of the TCR-complex or CD16, respectively, can be
enhanced through an NKG2D-mediated signal (Example 6).
[0021] Most surprisingly, however, NK- and T-cell cytotoxicity as
well as T-cell priming could be even enhanced by NKG2D-directed
antibody molecules, which by themselves did not induce any
substantial redirected lysis (Examples 5 and 6).
[0022] Thus, multifunctional NKG2D-directed polypeptides of the
invention with different properties of recruiting cytotoxic
lymphocytes may be advantageously selected for different purposes.
For example, if pure immunomodulation is required, NKG2D-directed
molecules may be preferred, which do not induce re-directed lysis
by themselves. However, target cell elimination may be more
pronounced when multifunctional NKG2D-directed polypeptides are
used that directly trigger lymphocyte cytotoxicity. Moreover,
multifunctional NKG2D-directed polypeptides, which differentially
recruit CD8.sup.+ T-cells and NK-cells, may be also preferable for
certain applications.
[0023] In a preferred embodiment of the method of the present
invention said binding site is the binding site of an
immunoglobulin chain.
[0024] In another preferred embodiment of the method of the present
invention said binding site is a natural NKG2D-ligand of said
receptor complex.
[0025] In a particularly preferred embodiment of the method of the
present invention said natural NKG2D-ligand is selected from the
group consisting of MIC-A, MIC-B, ULBP1 and ULBP2.
[0026] In another preferred embodiment of the method of the present
invention said binding site specifically recognizes an
extracellular epitope of NKG2D or of DAP10.
[0027] Further, in a preferred embodiment of the method of the
present invention said receptor or ligand function is an antigen
binding site of antibodies or fragments or derivatives thereof
against tumor associated antigens, antigens of infective agents or
surface markers of sub-populations of cells such as differentiation
antigens (CD antigens), natural ligands or receptors or fragments
thereof or modifications thereof that interact with tumor
associated antigens or surface markers, preferably heregulins,
binding to the tumor associated antigens erbB-2, -3 and -4, CD4
that interacts with gp 120 of HIV infected cells or melanocyte
stimulating hormone (MSH) that binds to the MSH receptor on
melanocytes and tumors derived therefrom (maligned melanomes) or
chemokines binding to corresponding chemokine receptors, or MHC
molecules or fragments thereof complexed with peptides that bind to
T-cell receptors of predefined specificity and thus recognize
certain T-cell sub-populations or antigen binding sites of T-cell
receptors that specifically interact with predefined MHC peptide
complexes, or NKp46 which interacts with haemagglutinin (HA) of
influenza virus.
[0028] Previous reports indicated that haemagglutinin of Influenza
virus can enhance lysis of virus-infected target cells by NK cells
as well as activate NK cells directly (Trinchiere, Adv. Immunol. 47
(1989), 187-376 and Alsheikhly, Scand J Immunol., 17 (1983), 129-38
and Alsheikhly, Scand J Immunol. 22 (1985), 529-38). It was shown
very recently that a fusion protein consisting of the extracellular
domain of NKp46 and the Fc portion of immunoglobulin (Ig) directly
bound to haemagglutinin-neuramini- dase (HN) glycoprotein expressed
on the cell surface of transiently transfected 293 cells
(Mandelboim, Nature 409 (2001), 1055-60). Addition of NK Gal cells,
a NK line derived from healthy donor peripheral blood lymphocytes
induced lysis of HN-transfected 293T cells at least four fold more
efficiently than of non-transfected cells (Mandelboim, Nature 409
(2001), 1055-60). The same results were obtained for Influenza
virus infected target cells. These data indicate that there is a
direct interaction between NKp46 and haemagglutinin, and, further,
demonstrate that the mechanism for elimination of Influenza virus
infected cells by NK cells is due to the interaction of
haemagglutinin (HA) exposed on virus infected cells and NKp46
expressed on the surface of NK cells.
[0029] Said receptor or ligand function which is capable of binding
to haemagglutinin (HA) of influenza virus is for example derived
from monoclonal antibodies like:
[0030] a) monoclonal antibody IIB4 binding to residues 155, 159,
188, 189, 193, 198, 199, 201 of influenza A virus strains H3
(Kostolansky, J Gen 81 (2000), 1727-35).
[0031] b) monoclonal antibody LMBH6 derived from mice sequentially
immunized with bromelain-cleaved haemagglutinin (BHA) from
influenza virus A/Aichi/2/68, A/Victoria/3/75 and
A/Philippines/2/82 (all H3N2) which recognizes HA of H3N2 influenza
A strains (Vanlandschoot, J. Gen. Virol. 79 (1998), 1781-91).
[0032] c) monoclonal antibody (MoAb) C179 directed to the stem
region of HA-H2 (Lipatov, Acta Virol. 41(1997), 337-40).
[0033] In this embodiment, said second domain represents in one
preferred embodiment an antigen which is the extracellular part of
a surface molecule on cells that are involved in pathologic
processes of human diseases like e.g. cancer, viral infections or
autoimmune conditions. Elimination or functional silencing of such
target cells may be facilitated by in vivo application of the
bifunctional molecules of the invention, thus providing therapeutic
benefit.
[0034] "Fragments" of said antibodies retain the binding
specificity of the complete antibodies and include Fab,
F(ab').sub.2 and Fv fragments. "Derivatives" of said antibodies
also retain the binding specificity and include scFv fragments. For
further information, see Marlow and Lane, "Antibodies, A Laboratory
Mammal" CSH Press, Cold Spring Harbor 1988.
[0035] Human cancer diseases may be, for example, cancers like
mamma carcinoma, breast cancer, colon carcinoma, pancreas
carcinoma, ovarian carcinoma, renal cell and cervix carcinoma,
melanoma, small cell lung cancer (SCLC), head and neck cancer,
gastric carcinoma, rhabdomyosarcoma, prostate carcinoma, folicular
Non-Hodgkin lymphoma (NHL), B cell lymphoma, multiple myeloma, T
and B cell leukemias and Hodgkin lymphoma.
[0036] Tumor associated antigens comprise pan-carcinoma antigens
like CEA (Sundblad Hum. Pathol. 27, (1996) 297-301, Ilantzis Lab.
Invest. 76(1997), 703-16), EGFR type I (Nouri, Int. J. Mol. Med. 6
(2000), 495-500) and EpCAM (17-1A/KSA/GA733-2, Balzar J. Mol. Med.
77 (1999), 699-712). EGFR type I is especially overexpressed in
glioma and EpCAM in colon carcinoma, MRD (minimal residual disease)
and colon carcinoma. EGFR type II (Her-2, ErbB2, Sugano Int. J.
Cancer 89 (2000), 329-36) is upregulated in mamma carcinoma and
TAG-72 glycoprotein (sTN antigen, Kathan Arch. Pathol. Lab. Med.
124 (2000), 234-9) was found to be expressed in breast cancer. EGFR
deletion neoepitope might also play a role as tumor associated
antigen (Sampson Proc. Natl. Acad. Sci. USA 97 (2000), 7503-8). The
antigens A33 (Ritter Biochem. Biophys. Res. Commun. 236 (1997),
682-6), Lewis-Y (DiCarlo Oncol. Rep. 8 (2001), 387-92), Cora
Antigen (CEA-related Cell Adhesion Molecule CEACAM 6, CD66c,
NCA-90, Kinugasa Int. J. Cancer 76 (1998), 148-53) and MUC-1
(Mucin) are associated with colon carcinoma (Iida Oncol. Res. 10
(1998), 407-14). Thomsen-Friedenreich-antigen (TF,
Gal1.beta.-3GalNAc.alpha.1-O-Thr/Ser) is not only found in colon
carcinoma (Baldus Cancer 82 (1998), 1019-27) but also in breast
cancer (Glinsky Cancer. Res. 60 (2000), 2584-8). Overexpression of
Ly-6 (Eshel J. Biol. Chem. 275 (2000), 12833-40) and desmoglein 4
in head and neck cancer and of E-cadherin neoepitope in castric
carcinoma was described (Fukudome Int. J. Cancer 88 (2000),
579-83). Prostate-specific membrane antigen (PSMA, Lapidus Prostate
45 (2000), 350-4), prostate stem cell antigen (PSCA, Gu Oncogene
191 (2000) 288-96) and STEAP (Hubert, Proc Natl Acad Sci USA 96
(1999), 14523-8) were associated with prostate cancer. The alpha
and gamma subunit of the fetal type acetylcholine receptor (AChR)
are specific immunohistochemical markers for rhabdomyosarcoma (RMS,
Gattenlohner Diagn. Mol. Pathol. 3 (1998), 129-34).
[0037] Association of CD20 with follicular non-Hodgkin lymphoma
(Yatabe Blood 95 (2000), 2253-61, Vose Oncology (Huntingt) 2 (2001)
141-7), of CD19 with B-cell lymphoma (Kroft Am. J. Clin. Pathol.
115 (2001), 385-95), of Wue-1 plasma cell antigen with multiple
myeloma (Greiner Virchows Arch 437 (2000), 372-9), of CD22 with B
cell leukemia (drena Am. J. Hematol. 64 (2000), 275-81), of CD7
with T-cell leukemia (Porwit-MacDonald Leukemia 14 (2000), 816-25)
and CD25 with certain T and B cell leukemias had been described (Wu
Arch. Pathol. Lab. Med. 124 (2000), 1710-3). CD30 was associated
with Hodgkin-lymphoma (Mir Blood 96 (2000), 4307-12). Expression of
melanoma chondroitin sulfate proteoglycan (MCSP, Eisenmann Nat.
Cell. Biol. 8 (1999), 507-13) and ganglioside GD3 was observed in
melanoma (Welte Exp Dermatol 2 (1997), 64-9), while GD3 was also
found in small lung cell cancer (SCLC, Brezicka Lung Cancer 1
(2000), 29-36). Expression of ganglioside GD2 was also upregulated
in SCLC and in neuroblastoma (Cheresh et al. Cancer Res. 10 (1986),
5112-8). Ovarian carcinoma was associated with Muellerian
Inhibitory Substance (MIS) receptor type II (Masiakos Clin. Cancer
Res. 11 (1999), 3488-99) and renal as well as cervix carcinoma with
expression of carboanhydrase 9 (MN/CAIX, Grabmaier Int. J. Cancer
85 (2000) 865-70). Elevated expression levels of CA 19-9 were found
in pancreas carcinoma (Nazli Hepatogastroenterology 47 (2000),
1750-2).
[0038] In a most preferred embodiment of the method of the present
invention said tumor-associated antigen is selected from the group
consisting of Lewis Y, CEA, Muc-1, erbB-2, -3 and -4, Ep-CAM,
E-cadherin neoepitope, EGF-receptor (e.g. EGFR type I or EGFR type
II), EGFR deletion neoepitope, CA19-9, Muc-1, LeY, TF-, Tn- and
sTn-antigen, TAG-72, PSMA, STEAP, Cora antigen, CD7, CD19 and CD20,
CD22, CD25, Ig-.alpha. and Ig-.beta., A33 and G250, CD30, MCSP and
gp100, CD44-v6, MT-MMPS, (MIS) receptor type II, carboanhydrase 9,
F19-antigen, Ly6, desmoglein 4, PSCA, Wue-1, GD2 and GD3 as well as
TM4SF-antigens (CD63, L6, CO-29, SAS) or the alpha and gamma
subunit of the fetal type acetylcholinreceptor (AChR).
[0039] Influenza A, B and C all have a segmented genome, but only
certain influenza A subtypes and influenza B cause severe disease
in humans. The two major proteins of influenza are the surface
glycoproteins-haemaggluti- nin (HA) and neuraminidase (NA).
Haemagglutinin (HA) is involved in the binding and membrane fusion
of virus particles to host cells receptors and represents the major
target for neutralizing antibodies. Infectivity of influenza
depends on the cleavage of HA by specific host proteases, whereas
NA is involved in the release of progeny virions from the cell. In
birds, the natural hosts of influenza, the virus causes
gastrointestinal infection and is transmitted via the faeco-oral
route. In mammals, replication of influenza subtypes appears
restricted to respiratory epithelial cells but systemic
complications can occur.
[0040] Rubella virus (RV) is the causative agent of the disease
known as measles. Rubella is predominantly a childhood disease and
is endemic throughout the world. Natural infections of rubella
occur only in humans and are generally mild but complications like
polyathralgia can occur in adults. RV infection of women during the
first trimester of pregnancy can induce a spectrum of congenital
defects in the newborn, known as congenital rubella syndrome (CRS).
The pathway whereby RV infection leads to teratogenesis has not
been elucidated. Cytopathology in infected fetal tissues suggests
necrosis and/or apoptosis as well as inhibition of cell division of
precursor cells involved in organogenesis. Rubella virus (RV)
virions contain two glycosylated membrane proteins, E1 and E2, that
exist as a heterodimer and form the viral spike complexes on the
virion surface. Formation of an E1-E2 heterodimer is essential for
intracellular transport and cell surface expression of both E1 and
E2 (Yang, J. Virol. 72 (1998), 8747-8755). Glycoproteins E1 and E2
expressed on rubella virus infected cells represent target
molecules for binding of multifunctional polypeptides of the
invention.
[0041] Rabies is an important disease in wildlife and dog rabies is
still a major public health problem in many developing countries of
the world. Rabies virus is transmitted in saliva by animal bites.
Most recently bats were found to transmit rabies to humans, often
without known exposures. In its classic form, rabies is well
recognized, but in cases with a paralytic illness mimicking
Landre's Guillain-Barre syndrome diagnosis remains problematically.
After exposure rabies can be prevented in non-immunized patients by
local wound cleansing and application of rabies vaccine and human
rabies-specific immunoglobulins. Rabies glycoprotein RGP is a 505
amino acid type I transmembrane glycoprotein which is important in
the biology and pathogenesis of rabies virus infection. RGP also
stimulates the development of neutralizing antibodies by the host.
N-linked glycosylation is required for immunogenicity and cell
surface expression of RGP (Wojczyk, Biochemistry 34 (1995),
2599-2609). RGP of rabies virus expressed on the surface of
infected cells represents a target molecules for binding of
multifunctional polypeptides of the invention.
[0042] In another most preferred embodiment of the method of the
present invention said surface marker for an infected cell is
selected from the group consisting of viral envelope antigens, e.g.
of human retroviruses (HTLV I and II, HIV1 and 2) or human herpes
viruses (HSV1 and 2, CMV, EBV), haemagglutinin e.g. of influenza
virus (influenza A, B or C), glycoproteins E1 and E2 from rubella
virus or RGP of rabies virus.
[0043] In another preferred embodiment of the method of the present
invention said multifunctional polypeptide is a bi-specific
molecule, preferably a bi-specific antibody. For further
information about the construction and generation of
bi-specific-antibodies, see WO/00/06605.
[0044] In a particularly preferred embodiment of the method of the
present invention said multifunctional polypeptide is selected from
the group consisting of a synthetic, a chimeric and a humanized
antibody.
[0045] In a further preferred embodiment of the method of the
present invention said multifunctional polypeptide is a
single-chain.
[0046] In an additional preferred embodiment of the method of the
present invention said two domains are connected by a polypeptide
linker.
[0047] In another preferred embodiment of the method of the present
invention said first and/or second domain mimic or correspond to a
V.sub.H and V.sub.L region of a natural antibody. Examples of such
antibodies comprise human, murine, rat and camel antibodies;
antibodies derived from immortalized B-cells (e.g. hybridoma
cells), from in vitro section of combinatorial antibody libraries
(e.g. by plage display) or from Ig-transgenic mice.
[0048] In a further preferred embodiment of the method of the
present invention at least one of said domains is a single-chain
fragment of the variable region of said antibody.
[0049] In an additional preferred embodiment of the method of the
present invention said domains are ranged in the order
V.sub.LNKG2D-V.sub.HNKG2D-- V.sub.HTA-V.sub.L-TA, or
V.sub.L-TA-V.sub.HTA-V.sub.HNKG2D-V.sub.LNKG2D wherein the TA
represents a target antigen.
[0050] In a particularly preferred embodiment of the method of the
present invention said tumor-associated antigen is selected from
the group consisting of Lewis Y, CEA, Muc-1, erbB-2, -3 and -4,
Ep-CAM, E-cadherin neoepitope, EGF-receptor (e.g. EGFR type I or
EGFR type II), EGFR deletion neoepitope, CA19-9, Muc-1, LeY, TF-,
Tn- and sTn-antigen, TAG-72, PSMA, STEAP, Cora antigen, CD7, CD19
and CD20, CD22, CD25, Ig-.alpha. and Ig-.beta., A33 and G250, CD30,
MCSP and gp100, CD44-v6, MT-MMPs, (MIS) receptor type II,
carboanhydrase 9, F19-antigen, Ly6, desmoglein 4, PSCA, Wue-1, GD2
and GD3 as well as TM4SF-antigens (CD63, L6, CO-29, SAS) or the
alpha and gamma subunit of the fetal type acetylcholinreceptor
(AChR).
[0051] In another particularly preferred embodiment of the method
of the present invention said polypeptide linker comprises a
plurality of glycine, serine and/or alanine residues.
[0052] In one further particularly preferred embodiment of the
method of the present invention said polypeptide linker comprises a
plurality of consecutive copies of an amino acid sequence.
[0053] Furthermore, in a particularly preferred embodiment of the
method of the present invention said polypeptide linker comprises 1
to 5, 5 to 10 or 10 to 15 amino acid residues.
[0054] In a most preferred embodiment of the method of the present
invention said polypeptide linker comprises the amino acid sequence
Gly-Gly-Gly-Gly-Ser.
[0055] In a further preferred embodiment of the method of the
present invention said multifunctional polypeptide comprises at
least one further domain. Target cell specific immune responses may
be further supported by combining the bifunctional molecules of the
invention with agents that confer costimulatory or coactivating
properties on the target cells.
[0056] In one alternative of the combination with additional
agents, the molecules of the invention may themselves be equipped
with additional functional domains, that may be joined e.g. through
another amino acid linker. These additional domains may e.g.
mediate CD28- or CD137-engagement (see below). Furthermore, it is
envisaged that derivatives of the bifunctional molecules of the
invention may be constructed that contain more than one additional
functional domain.
[0057] Alternatively, the molecules of the invention may be
combined with more than one additional agent in a composition e.g.
with one of said molecules engaging CD28 and another one engaging
CD137.
[0058] These agents referred to above may e.g. consist of a binding
site specifically recognizing the target cells and the
extracellular domain of B7-1 (CD80) or B7-2 (CD86) that interact
with CD28 on T- and NK-cells. Alternatively, B7-1 or B7-2 may be
replaced by the binding site of a CD28-specific antibody. On
T-lymphocytes CD28 acts as costimulatory molecule, which is
absolutely required in order to mediate the so-called second signal
during primary T cell activation through antigen specific
TCR-engagement (=first signal). On NK cells CD28 contributes to the
induction of cytotoxicity against target cells expressing CD28
ligands (Chambers (1996) Immunity 5: 311). Other agents that may be
advantageously combined with the bifunctional molecules of the
invention may consist of a binding site specifically recognizing
the target cells and the binding site of a CD137-specific antibody
or the extracellular part of the CD137-ligand.
[0059] In a most preferred embodiment of the method of the present
invention said further domain is linked by covalent or non-covalent
bonds.
[0060] In another most preferred embodiment of the method of the
present invention said at least one further domain comprises an
effector molecule having a conformation suitable for biological
activity, capable of sequestering an ion or selective binding to a
solid support or to a preselected determinant.
[0061] In a further most preferred embodiment of the method of the
present invention said further domain confers a co-stimulatory
and/or a co-activating function.
[0062] In a particularly preferred embodiment of the method of the
present invention said co-stimulatory function is mediated by a
CD28-ligand or a CD137-ligand.
[0063] In a further particularly preferred embodiment of the method
of the present invention said CD28-ligand or CD137-ligand is B7-1
(CD80), B7-2 (CD86), an aptamer or an antibody or a functional
fragment or a functional derivative thereof.
[0064] The term "functional fragment" of an antibody is defined as
a fragment of an antibody that retains the binding specificity of
said antibody (see, for example, Harlow and Lane, "Antibodies, A
Laboratory Manual" LSH Press, Cold Spring Harbor, 1988). Examples
of such fragments are Fab and F(ab).sub.2 fragment. "Functional
derivatives" of said antibodies retain or essentially retain the
binding specificity of said antibody. An example of said derivative
is an scFv Fragment.
[0065] The invention also relates to a polynucleotide which upon
expression encodes a multifunctional polypeptide and/or functional
parts of a multifunctional polypeptide of the invention. The term
"functional part" is defined in accordance with the invention as to
the part that confers the specific function of the first, second or
any further domain of a multifunctional polypeptide construct of
the invention. The polynucleotide may be DNA, RNA or a derivative
thereof such as PNA. Preferably, said polynucleotide is DNA.
[0066] Furthermore, the invention relates to a vector comprising
the polynucleotide of the present invention.
[0067] Many suitable vectors are known to those skilled in
molecular biology, the choice of which would depend on the function
desired and include plasmids, cosmids, viruses, bacteriophages and
other vectors used conventionally in genetic engineering. Methods
which are well known to those skilled in the art can be used to
construct various plasmids and vectors; see, for example, the
techniques described in Sambrook, Molecular Cloning A Laboratory
Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel,
Current Protocols in Molecular Biology, Green Publishing Associates
and Wiley Interscience, N.Y. (1989), (1994). The vectors of the
invention can be reconstituted into liposomes for delivery to
target cells.
[0068] The vector may be, for example, a phage, plasmid, viral, or
retroviral vector. Retroviral vectors may be replication competent
or replication defective. In the latter case, viral propagation
generally will occur only in complementing host cells.
Polynucleotides may be joined to a vector containing a selectable
marker for propagation in a host. Generally, a plasmid vector is
introduced in a precipitate, such as a calcium phosphate
precipitate, or in a complex with a charged lipid. If the vector is
a virus, it may be packaged in vitro using an appropriate packaging
cell line and then transduced into host cells.
[0069] The polynucleotide insert should be operatively linked to an
appropriate promoter, such as the phage lambda PL promoter, the E.
coli lac, trp, phoA and tac promoters, the SV40 early and late
promoters and promoters of retroviral LTRs, to name a few. Other
suitable promoters will be known to the skilled artisan. The
expression constructs will further contain sites for transcription
initiation, termination, and, in the transcribed region, a ribosome
binding site for translation. The coding portion of the transcripts
expressed by the constructs will preferably include a translation
initiating codon at the beginning and a termination codon (UAA, UGA
or UAG) appropriately positioned at the end of the polypeptide to
be translated.
[0070] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase, G418 or neomycin resistance for eukaryotic cell culture
and tetracycline, kanamycin or ampicillin resistance genes for
culturing in E. coli and other bacteria. Representative examples of
appropriate hosts include, but are not limited to, bacterial cells,
such as E. coli, Streptomyces and Salmonella typhimurium cells;
fungal cells, such as yeast cells; insect cells such as Drosophila
S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293,
and Bowes melanoma cells; and plant cells. Appropriate culture
mediums and conditions for the above-described host cells are known
in the art.
[0071] Among vectors preferred for use in bacteria include pQE70,
pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors,
Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from
Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3,
pDR540, pRIT5 available from Pharmacia Biotech, Inc.
[0072] Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,
pOG44, pXTI and pSG available from Stratagene; and pSVK3, pBPV,
pMSG and pSVL available from Pharmacia. In general, typical cloning
vectors include pBscpt sk, PGEM, pUC9, pBR322 and pGBT9. Typical
expression vectors include pTRE, pCAL-n-EK, pESP-1, pOP13CAT. Other
suitable vectors will be readily apparent to the skilled
artisan.
[0073] Furthermore, one could use, e.g., a mammalian cell that
already comprises in its genome a nucleic acid molecule encoding a
polypeptide as described above, but does not express the same or
not in an appropriate manner due to, e.g., a weak promoter, and
introduce into the mammalian cell a regulatory sequence such as a
strong promoter in close proximity to the endogenous nucleic acid
molecule encoding said polypeptide so as to induce expression of
the same.
[0074] In this context the term "regulatory sequence" denotes a
nucleic acid molecule that can be used to increase the expression
of the polypeptide, due to its integration into the genome of a
cell in close proximity to the encoding gene. Such regulatory
sequences comprise promoters, enhancers, inactivated silencer
intron sequences, 3'UTR and/or 5'UTR coding regions, protein and/or
RNA stabilizing elements, nucleic acid molecules encoding a
regulatory protein, e.g., a transcription factor, capable of
inducing or triggering the expression of the gene or other gene
expression control elements which are known to activate gene
expression and/or increase the amount of the gene product. The
introduction of said regulatory sequence leads to increase and/or
induction of expression of polypeptides, resulting in the end in an
increased amount of polypeptides in the cell. Thus, the present
invention is aiming at providing de novo and/or increased
expression of polypeptides.
[0075] The invention further relates to a cell transfected with the
polynucleotide of the present invention.
[0076] The cell of the invention may be a eukaryotic (e.g. yeast,
insect or mammalian) or prokaryotic cell. Most preferably, the cell
of the invention is a mammalian such as a human cell which may be a
member of a cell line e.g. CHO-cells, COS, 293, or Bowes melanoma
cells.
[0077] Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection, or other methods. Such
methods are described in many standard laboratory manuals, such as
Davis, Basic Methods In Molecular Biology (1986). It is
specifically contemplated that polypeptides may in fact be
expressed by a host cell lacking a recombinant vector.
[0078] The present invention further provides nucleic acid
molecules comprising a polynucleotide encoding upon expression a
multifunctional polypeptide and/or functional parts of a
multifunctional polypeptide of the invention as described herein
and in the appended examples. The nucleic acid sequence of two
different fragments of human NKG2D from nucleotides (nt) 64 to 462
and from (nt) 123 to 462 corresponding to amino acid sequences SEQ
ID 3 and 4 were PCR-amplified from the cDNA-template shown in FIG.
1. The resulting plasmids VV1-NKG2-D (nt 64-462) and VV1-NKG2-D (nt
123-462) were used to immunize three 6 to 8 weeks old BALB/c mice
as mentioned in the appended examples. Resulting lymphocytes were
fused with SP2/0 mouse myeloma cells (American Tissue Type
Collection, USA) in order to perform hybridoma selection as
indicated in the appended examples. Three hybridomas designated
11B2, 8G7 and 6E5 were shown to produce monoclonal antibodies
reactive with native NKG2D on the surface of both human CD8.sup.+
T-lymphocytes and NK-cells (for further information see appended
examples). Supernatants of the subclones 11B2D10, 8G7C10 and 6E5A7
were shown to react with NKG2-D on CD56.sup.+ NK- and CD8.sup.+ T
cells (as demonstrated in the appended examples). These subclones
were deposited, at the DSMZ-Deutsche Sammlung von Mikroorganismen
und Zellkulturen GmbH, Mascheroder Weg 1b, 38124 Braunschweig,
Germany on Mar. 23, 2001, in accordance with the provisions of the
Budapest Treaty and given accession number DSM ______, DSM ______
and DSM ______, respectively.
[0079] Additionally, the invention relates to a method for the
preparation of the multifunctional polypeptide and/or parts of the
multifunctional polypeptide of the invention comprising culturing a
cell of the present invention and isolating said multifunctional
polypeptide or functional parts thereof from the culture as
described for example by Mack, 1995, PNAS, 92, 7021.
[0080] Polypeptides can be recovered and purified from recombinant
cell cultures by well-known methods including ammonium sulfate or
ethanol precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography ("HPLC") is
employed for purification.
[0081] Depending upon the host employed in a recombinant production
procedure, the polypeptides may be glycosylated or may be
non-glycosylated. In addition, polypeptides may also include an
initial (modified) methionine residue, in some cases as a result of
host-mediated processes. Thus, it is well known in the art that the
N-terminal methionine encoded by the translation initiation codon
generally is removed with high efficiency from any protein after
translation in all eukaryotic cells. While the N-terminal
methionine on most proteins also is efficiently removed in most
prokaryotes, for some proteins, this prokaryotic removal process is
inefficient, depending on the nature of the amino acid to which the
N-terminal methionine is covalently linked.
[0082] It is also to be understood that the proteins can be
expressed in a cell free system using for example in vitro
translation assays known in the art.
[0083] The term "expression" means the production of a protein or
nucleotide sequence in the cell. However, said term also includes
expression of the protein in a cell-free system. It includes
transcription into an RNA product, post-transcriptional
modification and/or translation to a protein product or polypeptide
from a DNA encoding that product, as well as possible
post-translational modifications; see also supra. Depending on the
specific constructs and conditions used, the protein may be
recovered from the cells, from the culture medium or from both. The
terms "protein" and "polypeptide" used in this application are
interchangeable. "Polypeptide" refers to a polymer of amino acids
(amino acid sequence) and does not refer to a specific length of
the molecule. Thus peptides and oligopeptides are included within
the definition of polypeptide. This term does also refer to or
include post-translational modifications of the polypeptide, for
example, glycosylations, acetylations, phosphorylations and the
like; see also supra. Included within the definition are, for
example, polypeptides containing one or more analogs of an amino
acid (including, for example, unnatural amino acids, etc.),
polypeptides with substituted linkages, as well as other
modifications known in the art, both naturally occurring and
non-naturally occurring. For example, it is well known by the
person skilled in the art that it is not only possible to express a
native protein but also to express the protein as fusion
polypeptides or to add signal sequences directing the protein to
specific compartments of the host cell, e.g., ensuring secretion of
the protein into the culture medium, etc. The protein of the
invention may also be expressed as a recombinant protein with one
(polypeptide) or more additional polypeptide domains added to
facilitate protein purification. Such purification facilitating
domains include, but are not limited to, metal chelating peptides
such as histidine-tryptophan modules that allow purification
immobilized metals, protein A domains that allow purification on
immobilized immunoglobulin, and the domain utilized in the FLAGS
extension/affinity purification system (Immunex Corp, Seattle,
Wash.). The inclusion of a cleavable linker sequences such as
Factor XA or enterokinase (Invitrogen, San Diego, Calif.) between
the purification domain and the protein of interest is useful to
facilitate purification. One such expression vector provides for
expression of a fusion protein compromising a cell cycle
interacting protein and contains nucleic acid encoding 6 histidine
residues followed by thioredoxin and an enterokinase cleavage site.
The histidine residues facilitate purification on IMIAC
(immobilized metal ion affinity chromatography as described in
Porath, Protein Expression and Purification 3 (1992), 263-281)
while the enterokinase cleavage site provides a means for purifying
the protein from the fusion protein. In addition to recombinant
production, fragments of the protein of the invention may be
produced by direct peptide synthesis using solid-phase techniques
(cf Stewart et al (1969) Solid Phase Peptide Synthesis, W H Freeman
Co, San Francisco; Merrifield, J. Am. Chem. Soc. 85 (1963),
2149-2154). In vitro protein synthesis may be performed using
manual techniques or by automation. Automated synthesis may be
achieved, for example, using Applied Biosystems 431A Peptide
Synthesizer (Perkin Elmer, Foster City, Calif.) in accordance with
the instructions provided by the manufacturer. Various fragments of
the polypeptide of the invention may be chemically synthesized
and/or modified separately and combined using chemical methods to
produce the full length molecule. Once expressed or synthesized,
the protein of the present invention can be purified according to
standard procedures of the art, including ammonium sulfate
precipitation, affinity columns, column chromatography, gel
electrophoresis and the like; see, Scopes, "Protein Purification",
Springer-Verlag, N.Y. (1982). Substantially pure proteins of at
least about 90 to 95% homogeneity are preferred, and 98 to 99% or
more homogeneity are most preferred, for pharmaceutical uses. Once
purified, partially or to homogeneity as desired, the proteins may
then be used therapeutically (including extracorporeally) or in
developing and performing assay procedures.
[0084] The invention also relates to a composition comprising the
polypeptide of the present invention, the polynucleotide of the
invention or the vector of the present invention.
[0085] In a preferred embodiment of the composition of the present
invention said composition further comprises a molecule conferring
a co-stimulatory and/or co-activating function.
[0086] In this embodiment, the composition may comprise a
multifunctional polypeptide that comprises or does not comprise
said further domain as defined herein above. If the multifunctional
polypeptide comprises a further domain that confers co-stimulatory
and/or co-activating function, then said further molecule comprised
in the composition of the invention may have the same or a
different co-stimulatory and/or co-activating function.
[0087] In said composition, the comprised ingredients are packaged
together as separately in one or more containers such as vials,
preferably under sterile conditions, optionally in buffers or
aqueous solutions, some of which are further specified herein
below.
[0088] In a particularly preferred embodiment of the composition of
the present invention said co-stimulatory function is mediated by a
CD28-ligand or a CD137-ligand.
[0089] In another particularly preferred embodiment of the
composition of the present invention said CD28-ligand or
CD137-ligand is B7-1 (CD80), B7-2 (CD86), an aptamer or an antibody
or a functional fragment or a functional derivative thereof.
[0090] In a further preferred embodiment of the composition of the
present invention said composition is a pharmaceutical composition
optionally further comprising a pharmaceutically acceptable
carrier.
[0091] The compositions can also include, depending on the
formulation desired, pharmaceutically acceptable, usually sterile,
non-toxic carriers or diluents, which are defined as vehicles
commonly used to formulate pharmaceutical compositions for animal
or human administration. The diluent is selected so as not to
affect the biological activity of the combination. Examples of such
diluents are distilled water, physiological saline, Ringer's
solutions, dextrose solution, and Hank's solution. In addition, the
pharmaceutical composition or formulation may also include other
carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic
stabilizers and the like. A therapeutically effective dose refers
to that amount of protein or its antibodies, antagonists, or
inhibitors which ameliorate the symptoms or condition. Therapeutic
efficacy and toxicity of such compounds can be determined by
standard pharmaceutical procedures in cell cultures or experimental
animals, e.g., ED50 (the dose therapeutically effective in 50% of
the population) and LD50 (the dose lethal to 50% of the
population). The dose ratio between therapeutic and toxic effects
is the therapeutic index, and it can be expressed as the ratio,
LD50/ED50.
[0092] Further examples of suitable pharmaceutical carriers are
well known in the art and include phosphate buffered saline
solutions, water, emulsions, such as oilwater emulsions, various
types of wetting agents, sterile solutions etc. Compositions
comprising such carriers can be formulated by well known
conventional methods. These pharmaceutical compositions can be
administered to the subject at a suitable dose. Administration of
the suitable compositions may be effected by different ways, e.g.,
by intravenous, intraperitoneal, subcutaneous, intramuscular,
topical or intradermal administration. The dosage regimen will be
determined by the attending physician and clinical factors. As is
well known in the medical arts, dosages for any one patient depends
upon many factors, including the patient's size, body surface area,
age, the particular compound to be administered, sex, time and
route of administration, general health, and other drugs being
administered concurrently. A typical dose can be, for example, in
the range of 0.001 to 1000 .mu.g (or of nucleic acid for expression
or for inhibition of expression in this range); however, doses
below or above this exemplary range are envisioned, especially
considering the aforementioned factors. Generally, the regimen as a
regular administration of the pharmaceutical composition should be
in the range of 1 pg to 10 mg units per day. If the regimen is a
continuous infusion, it should also be in the range of 0,1 .mu.g to
10 mg units per kilogram of body weight per minute,
respectively.
[0093] The daily oral dosage regimen will preferably be from about
0.1 to about 80 mg/kg of total body weight, preferably from about
0.2 to 30 mg/kg, more preferably from about 0.5 mg to 15 mg. The
daily parenteral dosage regimen about 0.1 .mu.g/kg to about 100
mg/kg of total body weight, preferably from about 0.3 .mu.g/kg to
about 10 mg/kg, and more preferably from about 1 .mu.g/kg to 1
mg/kg. The daily topical dosage regimen will preferably be from 0.1
mg to 150 mg, administered one to four, preferably two to three
times daily. The daily inhalation dosage regimen will preferably be
from about 0.01 mg/kg to about 1 mg/kg per day.
[0094] Progress can be monitored by periodic assessment. Dosages
will vary but a preferred dosage for intravenous administration of
DNA is from approximately 10.sup.6 to 10.sup.12 copies of the DNA
molecule. DNA may also be administered directly to the target site,
e.g., by biolistic delivery to an internal or external target site
or by catheter to a site in an artery. The compositions comprising,
e.g., the polynucleotide, nucleic acid molecule, polypeptide,
antibody, compound drug, pro-drug or pharmaceutically acceptable
salts thereof may conveniently be administered by any of the routes
conventionally used for drug administration, for instance, orally,
topically, parenterally or by inhalation. Acceptable salts comprise
acetate, methylester, HCl, sulfate, chloride and the like. The
drugs may be administered in conventional dosage forms prepared by
combining the drugs with standard pharmaceutical carriers according
to conventional procedures. The drugs and pro-drugs identified and
obtained in accordance with the present invention may also be
administered in conventional dosages in combination with a known,
second therapeutically active compound. Such therapeutically active
compounds comprise, for example, those mentioned above. These
procedures may involve mixing, granulating and compressing or
dissolving the ingredients as appropriate to the desired
preparation. It will be appreciated that the form and character of
the pharmaceutically acceptable character or diluent is dictated by
the amount of active ingredient with which it is to be combined,
the route of administration and other well-known variables. The
carrier(s) must be "acceptable" in the sense of being compatible
with the other ingredients of the formulation and not deleterious
to the recipient thereof. The pharmaceutical carrier employed may
be, for example, either a solid or liquid. Exemplary of solid
carriers are lactose, terra alba, sucrose, talc, gelatin, agar,
pectin, acacia, magnesium stearate, stearic acid and the like.
Exemplary of liquid carriers are phosphate buffered saline
solution, syrup, oil such as peanut oil and olive oil, water,
emulsions, various types of wetting agents, sterile solutions and
the like. Similarly, the carrier or diluent may include time delay
material well known in the art, such as glyceryl mono-stearate or
glyceryl distearate alone or with a wax. A wide variety of
pharmaceutical forms can be employed. Thus, if a solid carrier is
used, the preparation can be tableted, placed in a hard gelatin
capsule in powder or pellet form or in the form of a troche or
lozenge. The amount of solid carrier will vary widely but
preferably will be from about 25 mg to about 1 g. When a liquid
carrier is used, the preparation will be in the form of a syrup,
emulsion, soft gelatin capsule, sterile injectable liquid such as
an ampule or nonaqueous liquid suspension.
[0095] The composition may be administered topically, that is by
non-systemic administration. This includes the application
externally to the epidermis or the buccal cavity and the
instillation of such a compound into the ear, eye and nose, such
that compound does not significantly enter the blood stream. In
contrast, systemic administration refers to oral, intravenous,
intraperitoneal and intramuscular administration.
[0096] Formulations suitable for topical administration include
liquid or semi-liquid preparations suitable for penetration through
the skin to the site of inflammation such as liniments, lotions,
creams, ointments or pastes, and drops suitable for administration
to the eye, ear or nose. The active ingredient may comprise, for
topical administration, from 0.001% to 10% w/w, for instance from
1% to 2% by weight of the formulation. It may however comprise as
much as 10% w/w but preferably will comprise less than 5% w/w, more
preferably from 0.1% to 1% w/w of the formulation.
[0097] Lotions according to the present invention include those
suitable for application to the skin or eye which are suitable, for
example, for use in UV protection. An eye lotion may comprise a
sterile aqueous solution optionally containing a bactericide and
may be prepared by methods similar to those for the preparation of
drops. Lotions or liniments for application to the skin may also
include an agent to hasten drying and to cool the skin, such as an
alcohol or acetone, and/or a moisturizer such as glycerol or an oil
such as castor oil or arachis oil.
[0098] Creams, ointments or pastes according to the present
invention are semi-solid formulations of the active ingredient for
external application. They may be made by mixing the active
ingredient in finely-divided or powdered form, alone or in solution
or suspension in an aqueous or non-aqueous fluid, with the aid of
suitable machinery, with a greasy or non-greasy base. The base may
comprise hydrocarbons such as hard, soft or liquid paraffin,
glycerol, beeswax, a metallic soap; a mucilage; an oil of natural
origin such as almond, corn, arachis, castor or olive oil; wool fat
or its derivatives or a fatty acid such as steric or oleic acid
together with an alcohol such as propylene glycol or a macrogel.
The formulation may incorporate any suitable surface active agent
such as an anionic, cationic or non-ionic surfactant such as a
sorbitan ester or a polyoxyethylene derivative thereof. Suspending
agents such as natural gums, cellulose derivatives or inorganic
materials such as silicaceous silicas, and other ingredients such
as lanolin, may also be included.
[0099] Drops according to the present invention may comprise
sterile aqueous or oily solutions or suspensions and may be
prepared by dissolving the active ingredient in a suitable aqueous
solution of a bactericidal and/or fungicidal agent and/or any other
suitable preservative, and preferably including a surface active
agent. The resulting solution may then be clarified by filtration,
transferred to a suitable container which is then sealed and
sterilized by autoclaving or maintaining at 98-100.degree. C. for
half an hour. Alternatively, the solution may be sterilized by
filtration and transferred to the container by an aseptic
technique. Examples of bactericidal and fungicidal agents suitable
for inclusion in the drops are phenylmercuric nitrate or acetate
(0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate
(0.01%). Suitable solvents for the preparation of an oily solution
include glycerol, diluted alcohol and propylene glycol.
[0100] The composition in accordance with the present invention may
be administered parenterally, that is by intravenous,
intramuscular, subcutaneous intranasal, intrarectal, intravaginal
or intraperitoneal administration. The subcutaneous and
intramuscular forms of parenteral administration are generally
preferred. Appropriate dosage forms for such administration may be
prepared by conventional techniques. The composition may also be
administered by inhalation, that is by intranasal and oral
inhalation administration. Appropriate dosage forms for such
administration, such as an aerosol formulation or a metered dose
inhaler, may be prepared by conventional techniques.
[0101] In a different preferred embodiment of the composition of
the present invention said composition is a diagnostic composition
optionally further comprising suitable means for detections.
[0102] Said means for detection comprise, for example, (a)
chromophore(s), (a) fluorexcent dye(s), (a) radionucleotide(s),
biotin or DIG. These labeling means may be coupled to nucleotide
analogues. Labeling of amplified cDNA can be performed as described
in the appended examples or as described, inter alia, in Spirin
(1999), Invest. Opthamol. Vis. Sci. 40, 3108-3115.
[0103] The present invention also relates to a use of the
multifunctional polypeptide of the present invention, the
polynucleotide of the present invention or the vector of the
present invention for the preparation of a pharmaceutical
composition for the treatment of cancer, infections and/or
autoimmune conditions, cancer, i.e. maligned (solid) tumors and
hematopoietic cancer forms (leukemias and lymphomas), benigne
tumors such as benigne hyperplasia of the prostate gland (BPH),
autonomous adenomes of the thyroid gland or of other endocrine
glands or adenomas of the colon; initial stages of the
malignancies, infectious diseases, caused by viruses, bacteria,
fungi, protozoa or helmints, auto immune diseases wherein the
elimination of the subpopulation of immune cells is desired that
causes the disease; prevention of transplant rejection or
allergies.
[0104] In a preferred embodiment of the use of the present
invention said infection is said infection is a viral, a bacterial
or a fungal infection, wherein said cancer is a head and neck
cancer, gastric cancer, oesaphagus cancer, stomach cancer,
colorectal cancer, coloncarcinoma, cancer of liver and intrahepatic
bile ducts, pancreatic cancer, lung cancer, small cell lung cancer,
cancer of the larynx, breast cancer, mamma carcinoma, malignant
melanoma, multiple myeloma, sarcomas, rhabdomyosarcoma, lymphomas,
folicular non-Hodgkin-lymphoma, leukemias, T- and B-cell-leukemias,
Hodgkin-lymphoma, B-cell lymphoma, ovarian cancer, cancer of the
uterus, cervical cancer, prostate cancer, genital cancer, renal
cancer, cancer of the testis, thyroid cancer, bladder cancer,
plasmacytoma or brain cancer or wherein said autoimmune condition
is ankylosing spondylitis, acute anterior uveitis, Goodpasture's
syndrome, Multiple sclerosis, Graves' disease, Myasthenia gravis,
Systemic lupus erythematosius, Insulin-dependent diabetes mellitus,
Rheumatoid arthritis, Pemphigus vulgaris, Hashimoto's thyroiditis
or autoimmune Hepatitis.
[0105] The present invention also relates to a use of the
polynucleotide of the present invention or the vector of the
present invention for the preparation of a composition for gene
therapy.
[0106] It is envisaged by the present invention that the various
polynucleotides and vectors encoding the above described
phosphotonin peptides or polypeptides are administered either alone
or in any combination using standard vectors and/or gene delivery
systems, and optionally together with a pharmaceutically acceptable
carrier or excipient. For example, the polynucleotide of the
invention can be used alone or as part of a vector to express the
(poly)peptide of the invention in cells, for, e.g., gene therapy or
diagnostics of diseases related to disorders referred to above. The
polynucleotides or vectors of the invention are introduced into the
cells which in turn produce the (poly)peptide. Subsequent to
administration, said polynucleotides or vectors may be stably
integrated into the genome of the subject. On the other hand, viral
vectors may be used which are specific for certain cells or tissues
and persist in said cells. Suitable pharmaceutical carriers and
excipients are well known in the art. The polynucleotides or
vectors prepared according to the invention can be used for the
prevention or treatment or delaying of different kinds of the
diseases referred to above.
[0107] In the above-described embodiments, the vector of the
present invention may preferably be a gene transfer or targeting
vector. Gene therapy, which is based on introducing therapeutic
genes, for example for vaccination into cells by ex-vivo or in-vivo
techniques is one of the most important applications of gene
transfer. Suitable vectors, methods or gene-delivering systems for
in-vitro or in-vivo gene therapy are described in the literature
and are known to the person skilled in the art; see, e.g.,
Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79
(1996), 911-919; Anderson, Science 256 (1992), 808-813, Isner,
Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995),
1077-1086; Onodua, Blood 91 (1998), 30-36; Verzelefti, Hum. Gene
Ther. 9 (1998), 2243-2251; Verma, Nature 389 (1997), 239-242;
Anderson, Nature 392 (Supp. 1998), 25-30; Wang, Gene Therapy 4
(1997), 393-400; Wang, Nature Medicine 2 (1996), 714-716; WO
94/29469; WO 97/00957; U.S. Pat. Nos. 5,580,859; 5,589,466;
4,394,448 or Schaper, Current Opinion in Biotechnology 7 (1996),
635-640, and references cited therein.
[0108] The polynucleotides and vectors of the invention may be
designed for direct introduction or for introduction via liposomes,
or viral vectors (e.g. adenoviral, retroviral) into the cell.
Preferably, said cell is a germ line cell, embryonic cell, or egg
cell or derived therefrom, most preferably said cell used for
introduction is a stem cell. As mentioned above, suitable gene
delivery systems may include liposomes, receptor-mediated delivery
systems, naked DNA, and viral vectors such as herpes viruses,
retroviruses, adenoviruses, and adeno-associated viruses, among
others. Delivery of nucleic acids to a specific site in the body
for gene therapy may also be accomplished using a biolistic
delivery system, such as that described by Williams (Proc. Natl.
Acad. Sci. USA 88 (1991), 2726-2729).
[0109] It is to be understood that the introduced polynucleotides
and vectors express the gene product after introduction into said
cell and preferably remain in this status during the lifetime of
said cell. For example, cell lines which stably express the
polynucleotide under the control of appropriate regulatory
sequences may be engineered according to methods well known to
those skilled in the art. Rather than using expression vectors
which contain viral origins of replication, host cells can be
transformed with the polynucleotide of the invention and a
selectable marker, either on the same or separate plasmids.
Following the introduction of foreign DNA, engineered cells may be
allowed to grow for 1-2 days in an enriched media, and then are
switched to a selective media. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows
for the selection of cells having stably integrated the plasmid
into their chromosomes and grow to form foci which in turn can be
cloned and expanded into cell lines. Such engineered cell lines are
also particularly useful in screening methods for the detection of
compounds involved in, e.g., activation or stimulation of phosphate
uptake.
[0110] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler, Cell
11 (1977), 223), hypoxanthine-guanine phosphoribosyltransferase
(Szybalska, Proc. NatI. Acad. Sci. USA 48 (1962), 2026), and
adenine phosphoribosyltransferase (Lowy, Cell 22 (1980), 817) in
tk.sup.-, hgprt.sup.- or aprt.sup.- cells, respectively. Also,
antimetabolite resistance can be used as the basis of selection for
dhfr, which confers resistance to methotrexate (Wigler, Proc. Natl.
Acad. Sci. USA 77 (1980), 3567; O'Hare, Proc. Natl. Acad. Sci. USA
78 (1981), 1527), gpt, which confers resistance to mycophenolic
acid (Mulligan, Proc. Natl. Acad. Sci. USA 78 (1981), 2072); neo,
which confers resistance to the aminoglycoside G-418
(Colberre-Garapin, J. Mol. Biol. 150 (1981), 1); hygro, which
confers resistance to hygromycin (Santerre, Gene 30 (1984), 147);
or puromycin (pat, puromycin N-acetyl transferase). Additional
selectable genes have been described, for example, trpB, which
allows cells to utilize indole in place of tryptophan; hisD, which
allows cells to utilize histinol in place of histidine (Hartman,
Proc. Natl. Acad. Sci. USA 85 (1988), 8047); and ODC (ornithine
decarboxylase) which confers resistance to the ornithine
decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO
(McConlogue, 1987, In: Current Communications in Molecular Biology,
Cold Spring Harbor Laboratory ed.).
[0111] The invention further relates to a method for the treatment
of cancer, infections or autoimmune conditions comprising
introducing the polypeptide of the present invention, the
polynucleotide of the present invention or the vector of the
present invention or the composition of the present invention into
a mammal affected by said malignancies or diseases.
[0112] Suitable routes and doses of administration etc. have been
discussed in connection with the pharmaceutical composition of the
invention herein above.
[0113] Furthermore, the present invention relates to a method for
delaying a pathological condition comprising introducing the
polypeptide of the present invention, the polynucleotide of the
invention or the vector of the present invention or the composition
of the present invention into a mammal affected by said
pathological condition.
[0114] In a preferred embodiment of one method of the present
invention said mammal is a human.
[0115] Finally, the invention relates to a kit comprising the
multifunctional polypeptide of the invention, the polynucleotide of
the present invention, the vector of the present invention, the
cell of the invention or the composition of the present
invention.
[0116] The components of the kit or the diagnostic composition of
the present invention may be packaged in containers such as vials,
optionally in buffers and/or solutions. If appropriate, one or more
of said components may be packaged in one and the same container.
Additionally or alternatively, one or more of said components may
be absorbed to a solid support such as, e.g., a nitrocellulose
filter or nylon membrane, or to the well of a microtitre-plate.
[0117] The figures show:
[0118] FIG. 1 shows the nucleotide and amino acid sequence of
soluble NKG2D containing a C-terminal histidine-tag. Restriction
sites used for cloning are shown at the beginning (EcoRI) and the
end (SalI) of the nucleotide sequence.
[0119] FIG. 2 shows the molecular design of an NKG2D-directed
bispecific single-chain antibody at the DNA level (panel A) and the
protein level (panel B). The mode of function of the bispecific
antibody is also shown in panel B.
[0120] FIG. 3: SDS-PAGE of bispecific single-chain antibody
anti-NKG2D (8R23).times.anti-EpCAM (4-7) (right lane); the left
lane shows a molecular weight marker.
[0121] FIG. 4: Expression vector encoding a secreted
carboxy-terminal fragment of human NKG2-D used for genetic
immunization.
[0122] The expression of the NKG2-D fragment from the vector shown
is controlled by the immediate-early promoter of the human
cytomegalovirus (CMV). The NKG2-D fragment consists of a leader
peptide which is derived from the murine immunoglobulin kappa light
chain, followed by a human myc epitope. The coding sequence of
NKG2-D is terminated by its cognate stop codon. BGH polyadenylation
site, bovine growth hormone polyadenylation site; amp, ampicillin
resistance gene; ColE1 origin, ColE1 origin of replication.
[0123] FIG. 5: Selection of hybridomas specifically binding to
NKG2-D-positive target cells.
[0124] The binding of three distinct monoclonal antibodies in
hybridoma supernatants 6E5, 8G7 and 11B2 to either CD8-positive T
cells (A) or to CD56-positive natural killer cells is shown by FACS
analysis. Abbreviations are 6E5: 6E5/A7, 8G7: 8G7/C10 and 11B2:
11B2/D10.
[0125] 10H9 is a control with a hybridoma supernatant lacking
NKG2-D binding activity. The various detection antibodies are
indicated in the Figure.
[0126] FIG. 6: Enhancing effect of a monoclonal antibody directed
against NKG2-D on priming of naive T cells.
[0127] Native T cells expressing the marker CD45RA (A) are found in
FACS scans in the upper left gate. Naive T cells were primed in the
presence of an EpCAM-expressing target cell line
(EpCAM/17-1A-transfected CHO cells) by a combination of a
B7-1.times.anti-EpCAM fusion protein and a single chain bispecific
anti-EpCAM.times.anti-CD3 molecule (B-E) in the absence (D and E)
or presence (B and C) of a monoclonal antibody against NKG2-D
called BAT221. Primed T cells expressing the marker CD45 RO appear
in the lower right gate. Numbers-give the percentage of primed,
previously native T cells. Fluorescence 1: FITC-labeled
anti-CD45RO; fluoresence 2: phycoerythrin-conjugated
anti-CD45RA.
[0128] FIG. 7: Enhancing effect of a monoclonal antibody directed
against NKG2-D on TNF production by T cells.
[0129] Nave T cells were primed in the presence of an
EpCAM-expressing target cell line (EpCAM/17-1A-transfected CHO
cells) by a combination of a B7-1.times.anti-EpCAM fusion protein
and increasing concentrations, as indicated, of a single chain
bispecific anti-EpCAM.times.anti-CD3 molecule. TNF production was
measured by a commercial TNF-.alpha.: ELISA in the presence (A) and
absence (B) of a monoclonal antibody against NKG2-D, called
BAT221.
[0130] FIG. 8: Cytotoxic activity of Melan A cells and NKL cells
redirected against P815 cells by several dilutions of the
supernatant of the NKG2D hybridoma BAT 221 in combination with the
monoclonal antibodies CD16 (5 .mu.g/ml) and CD3 (0,2 .mu.g/ml)
respectively. 200.000 NKL cells or 50000 Melan A cells were added
to 10.000 Chromium-51 labeled Kato III cells in the presence of the
diluted antibody in a total volume of 200 .mu.l. The backround
control (E+T) contains effector cells and target cells without an
antibody dilution. The microtiterplates were incubated for 4 h at
37.degree. C., 5% CO.sub.2. After the incubation period 50 .mu.l
supernatant were removed from each well and assayed for released
.sup.51Cr in a gamma counter.
[0131] FIG. 9: Detection of a specific immune response in mice
immunized with an expression vector encoding a secreted C-terminal
fragment of human NKG2-D.
[0132] Flowcytometric analysis of the binding activity of a 1:30
serum diliution of five immunized mice to human CD8.sup.+ T
lymphocytes and human NK cells. 200.000 mononucleated cells from
peripheral blood of a healthy donors were incubated with diluted
serum of the five mice. Bound murine antibody was detected by a
fluoresceine (FITC)-conjugated goat-anti-rat Ig (IgG+IgM) antibody
diluted 1:100 in PBS. Triple color fluorescence analysis was
carried out by applying a positive gate for CD8.sup.+ (Tricolor)
and a negative gate for CD16.sup.+ (PE) cells thus allowing the
detection of FITC-mediated fluorescence exclusively attributed to
CD8.sup.+-T-lymphocytes (phenotype: CD8.sup.+, CD16.sup.-) without
any contaminating signals from CD8.sup.+-NK-cells. Similarly,
triple color fluorescence analysis was carried out by applying a
positive gate for CD56.sup.+-(PE) and a negative gate for
CD3.sup.+-cells (tricolor) thus allowing the detection of
FITC-mediated fluorescence exclusively attributed to NK-cells
(phenotype: CD56.sup.+, CD3.sup.-) without any contaminating
signals from CD56.sup.+-T-lymphocytes. As negative control a
representative serum of an unimmunized mouse was used (preimmune
serum). Cells were analyzed by flowcytometry on a FACSscan (Becton
Dickinson).
[0133] FIG. 10: Design of the phagemid used for expression of
N-terminally blocked single chain antibodies in the periplasm of E.
coli.
[0134] P, bacterial promoter; ompA, leader sequence for periplasmic
transport; N2, surrogate N-terminal blocking domain; VH, variable
heavy chain domain of scFv; VL, variable light chain domain of
scFv; p53, tetramerization domain of transcription factor p53;
Flag-tag; influenza virus epitope tag. The positions of various
restriction enzyme sites are indicated on top. Essential coding
sequences are shown as black boxes.
[0135] FIG. 11: Detection of NKG2-D-specific, N-terminally blocked
single chain Fv fragments produced in the periplasm of E. coli.
[0136] In order to increase sensitivity, the binding avidity of
single chain Fv antibodies was increased by fusing the
tetramerization domain of the transcription factor p53 to the
carboxy terminus of N-terminally blocked scFvs. Tetramerized scFvs
were detected in periplamsic fractions by ELISA with soluble,
recombinant NKG2-D as capture and peroxidase-conjugated anti-FLAG
antibody for detection. ELISA signals of various clones are
depicted. All clones with signals>0.05 were analyzed
further.
[0137] FIG. 12: Transient expression and EpCAM binding of four
bispecific molecules targeting NKG2-D.
[0138] CHO/dhfr- cells were transiently transfected with expression
vectors encoding four different single chain bispecific molecules.
In A, a beta-galactosidase gene was transfected as negative
control. The various bispecific molecules are B, 3B10.times.P4-3;
C, 3B10.times.P4-14, D, 3B10.times.P5-2 and E, 3B10.times.P5-23.
Cell culture supernatants were harvested after 5 days and tested
for the expression of bispecific antibodies by FACS analysis for
EpCAM-specific binding to the human gastric carcinoam cell line
Kato III. Cell-bound bispecific molecules were detected by an
FITC-labeled sheep-anti-mouse antibody. FACS histogram blots are
shown.
[0139] FIG. 13: Characterization of two single chain bispecific
antibodies for NKG2-D specific binding in an ELISA.
[0140] The two bispecific antibodies 3B10.times.P4-3 and
3B10.times.P5-2 were transiently expressed in CHO cell culture
supernatants. Binding to coated soluble, recombinant NKG2-D was
tested by an ELISA using a peroxidase-conjugated anti-hexahistidine
antibody for detection of the hexahistidine-tagged bispecific
antibodies. Two different concentrations were tested. A, 1:1
dilution; B, 1:2 dil ution of culture supernatants. As a control,
binding of an EpCAM-specific 3B10.times.anti-CD3 bispecific
antibody was used. Values obtained for this non-specific control
were subtracted fom the readings shown.
[0141] FIG. 14: Cytotoxic activity of Melan A cells (A) and NKL
cells (B) redirected against EpCAM-positive Kato cells by the
bispecific 3B10.times.P4-3 antibody. 200.000 NKL cells or 50000
Melan A cells were added to 10.000 Chromium-51 labeled Kato III
cells in the presence of serveral dilutions of the bispecific
antibody in a total volume of 200 .mu.l. The background control
(E+T) contains effector cells and target cells without an antibody
dilution. The microtiterplates were incubated for 4 h at 37.degree.
C., 5% CO.sub.2. After the incubation period 50 .mu.l supernatant
were removed from each well and assayed for released .sup.51Cr in a
gamma counter.
[0142] FIG. 15: Specific target cell lysis by four single chain
antibodies recruiting peripheral blood mononuclear cells (PBMCs)
via NKG2-D.
[0143] Four bispecific antibodies all recognizing the EPCAM target
on the human gastric carcinoma cell line Kato III by a single chain
Fv derived from monoclonal antibody 3B10 were contructed from four
distinct scFvs specific for the NK/CD8-specific receptor NKG2-D.
Expression vectors encoding the four bispecific antibodies were
transfected for transient expression into CHO cells and
supernatants collected. Supernatants with secreted bispecific
antibodies at the indicated dilutions were tested in cytotox assays
for specific lysis of Kato IlIl cells in the presence of human
immune effector cells (PBMCs). In the absence of CHO supernatants,
no target cell lysis of Kato III cells was observed in the presence
of PBMCs. Data shown are the means of triplicate determinations.
Cytotoxic activity of PBMC redirected against EPCAM-positive Kato
cells by the bispecific antibodies 3B10.times.P4-3;
3B10.times.P4-14; 3B10.times.P5-2 and 3B10.times.P5-23 in several
dilutions. 200.000 PBMCs were added to 10.000 Chromium-51 labeled
Kato III cells in the presence of the diluted bispecific antibodies
in a total volume of 200 .mu.l. The negative control contains PBMCs
and target cells without an antibody dilution. The microtiterplates
were incubated for 4 h at 37.degree. C., 5% CO.sub.2. After the
incubation period 50 .mu.l supernatant were removed from each well
and assayed for released .sup.51Cr in a gamma counter.
[0144] FIG. 16: Compilation of sequences as depicted in the
appended examples. The nucleotide sequences are shown in the common
5'-3' orientation.
[0145] The Examples illustrate the invention.
EXAMPLE 1
[0146] Production of Recombinant NKG2D
[0147] To obtain the coding DNA-sequence of the extracellular
portion of the NKG2D-antigen, cDNA derived from the RNA of
peripheral blood mononuclear cells by reverse transcription was
used as template for a polymerase chain reaction (PCR).
[0148] Total RNA was prepared from peripheral blood mononuclear
cells which were separated from a whole-blood sample by
ficoll-density centrifugation following standard protocols (J. E.
Coligan, Wiley Intersience 1991).
[0149] The RNA preparation was performed using a commercially
available preparation kit (Quiagen) according to the instructions
of the manufacturer.
[0150] The cDNA-synthesis was carried out according to standard
protocols (Sambrock, Cold Spring Harbor Laboratory Press 1989,
second edition)
[0151] For the PCR, a pair of primers with the following sequences
was used:
[0152] Forward primer:
5'-AGGTGTACACTCCTTATTCAACCAAGAAGTTCAAATTCC-3' (SEQ ID 87)
[0153] Reverse primer: 5'-TCATCCGGACACAGTCCTTTGCATGCAGATG-3' (SEQ
ID 88)
[0154] In addition to the sequence hybridizing to the NKG2D
cDNA-template, the forward primer contains a BsrGI-site and the
reverse primer a BspEI-site to allow the cloning of the PCR
amplification product.
[0155] The product of the PCR-reaction was isolated by means of an
agarose-gel electrophoresis, purified using a commercially
available kit (Quiagen) according to the instructions of the
manufacturer, and then incubated with the restriction enzymes BsrGI
and BspEI using standard protocols (Sambrock, Cold Spring Harbor
Laboratory Press 1989, second edition). Afterwards a final
purification step was performed.
[0156] As shown in FIG. 1, the coding sequence of the NKG2D
extracellular domain was fused via BsrGI to a murine Ig-heavy chain
leader sequence; the BspEI-site was fused with an XmaI-site thus
joining the coding sequence of a poly-histidine tag followed by a
stop codon (SEQ ID 1 and 2).
[0157] The EcoRI/SalI-DNA fragment shown in FIG. 1 consisting of
the coding sequences of an N-terminal leader peptide, the NKG2D
extracellular domain and a C-terminal histidine-tag, was cloned
into the plasmid vector pFastBac1 also prepared by digestion with
the restriction enzymes EcoRI and SalI. This plasmid is part of the
Bac-to-Bac.RTM. Baculovirus expression system (Gibco BRL,
instructions of the manufacturer are available at the internet
site: http://www2.lifetech.com/catalog/techline- /molecular
biology/Manuals PPS/bac.pdf. Unless stated otherwise, all
procedures related to the Bac-to-Bac.RTM. Baculovirus expression
system, were carried out according to these instructions).
[0158] 1 ng DNA of a correct plasmid clone was then tranisformed
into DH10Bac competent cells (Bac-to-Bac.RTM. expression system).
This Escherichia coli strain already carries two other plasmids,
(i) a helper plasmid (pMON7124) providing Tn7 transposition
functions and (ii) a so-called bacmid (pMON 14272) which is a
baculovirus shuttle vector. After transformation of the third
plasmid into these cells the coding sequence inserted into
pfastBacl is transferred by transposition into the bacmid which
contains specific target sites for this transposition. That leads
to the destruction of a LacZ-coding sequence which offers the
possibility to select colonies with the recombinant bacmid by means
of a blue white selection on agar plates containing Bluo-gal, IPTG
and a combination of antibiotics according to the instructions of
the manufacturer.
[0159] White colonies containing the recombinant bacmid with the
soluble NKG2D sequence were selected and cultured over night. A
specific protocol provided by the manufacturer was used for the
preparation of bacmid-DNA from these overnight cultures.
[0160] The bacmid-DNA was then used to transfect SF9-insect cells
using CellFectin Reagent (Bac-to-Bac.RTM. expression system)
according to the instructions of the manufacturer. Three days after
transfection recombinant baculovirus in the culture supernatant of
the transfected cells was harvested. This supernatant is a low
titer (approximately 2.times.10.sup.7 plaque forming units (pfu)
per millilitre) low scale (2 ml) virus stock. (Instructions for
insect cell culture, propagation of baculoviruses and protein
expression in the baculovirus expression system are available at
the internet site: http://www.invitrogen.com/manuals.htm- l. Unless
stated otherwise, all procedures related to insect cell culture and
protein expression were carried out according to these
instructions). For protein expression a high titer and high scale
virus stock was required. To obtain such a virus stock the
following steps were performed:
[0161] Two 25 cm.sup.2 tissue culture flasks each seeded with
2.times.10.sup.6 SF9-cells were infected with 30 .mu.l of the
initial virus stock, respectively. After ten days the culture
supernatants were harvested as a low scale--high titer viral stock.
Then a 500 ml suspension culture of SF9-cells at a density of
2,0.times.10.sup.6 cells per milliliter was infected with 5 ml of
the second virus stock. Progression of the infection was monitored
by determination of the cell viability using the trypan-blue
exclusion method. At a cell viability below 10% the viral stock was
harvested and virus supernatant separated from cells by
centrifugation. The viral titer of this large scale stock had to be
determined. For this purpose SF-9 cells were seeded in a 96-well
tissue culture plate at a density of 10.sup.4 cells per well. A
total of 24 wells was each infected with one of the following
dilutions of the high titer stock: 10 .mu.l of a 1:10.sup.5
dilution per well, 10 .mu.l of a 1:10.sup.6 dilution per well and
10 .mu.l of a dilution per well. The volume had to be adjusted to
120 .mu.l per well. After 14 days viability of the cells was
determined by the trypan-blue exclusion assay. That dilution with a
balanced relation of wells with viable and non-viable cells allows
a sufficiently precise estimation of the viral titer which is
expected to be 1.times.10.sup.8 to 1.times.10.sup.9 pfu/ml.
[0162] The time course of protein expression was determined at MOIs
(multiplicity of infection) of 5 pfu and 10 pfu per cell in an
infection experiment with two suspension cultures of SF9 cells at
2,3.times.10.sup.6 cells/ml. Samples of the infected cultures were
drawn at 24, 48, 72 and 96 hours post infection. These samples were
analysed by western blot according to standard protocols. Soluble
NKG2D was detected with a peroxidase-conjugated anti-histidine-tag
antibody.
[0163] Thus, the optimal MOI and the optimal incubation time after
infection were used for large scale protein expression in multiple
suspension cultures of 500 ml culture volume.
[0164] Soluble NKG2D was purified from culture supernatants via its
C-terminal histidine tag by affinity chromatography using a
Ni-NTA-column as described by Mack (1995) Proc Natl Acad Sci USA
92: 7021.
EXAMPLE 2
[0165] Generation of Monoclonal Antibodies Against Native NKG2D on
Human Lymphocytes
[0166] Ten weeks old F1 mice from balb/c.times.C57black crossings
were immunized with the soluble extracellular domain of the antigen
NKG2D. The antigen was dissolved in 0.9% NaCl at a concentration of
100 .mu.g/ml. The solution was subsequently emulsified 1:2 with
complete Freund's adjuvants and 50 .mu.l were injected per mouse
intraperitonially. Mice received booster immunizations after 4, 8,
and 12 weeks in the same way, except that complete Freund's
adjuvants was replaced by incomplete Freund's adjuvants. Ten days
after the first booster immunization, blood samples were taken and
antibody serum titer against NKG2D antigen was tested by ELISA.
Serum titer was more than 1000 times higher in immunized than in
not immunized animals. Three days after the second boost, spleen
cells were fused with P3.times.63Ag8.653 cells (ATCC CRL-1580) to
generate hybridoma cell lines following standard protocols as
described in Current Protocols in Immunology (Coligan, Kruisbeek,
Margulies, Shevach and Strober, Wiley Interscience, 1992). After
PEG-fusion, cells were seeded at 100.000 cells per well in
microtiterplates and grown in 200 .mu.l RPMI 1640 medium
supplemented with 10% fetal bovine serum, 300 units/ml recombinant
human interleukin 6 and HAT-additive for selection. Culture
supernatants from densely grown wells were tested by the following
ELISA:
[0167] The wells of a 96 U-bottom plate (Nunc, maxisorb) were
coated overnight at 4.degree. C. with recombinant NKG2D-antigen at
a concentration of 5 .mu.g/ml. Coated-wells were washed three times
with washing buffer (0.1M NaCl, 0.05M Na2HPO4 pH 7.3, 0.05% Tween
20, 0.05% NaN3) and subsequently blocked through incubation for one
hour at room temperature with 200 pl/well of 2% skimmed milk powder
suspended in washing buffer. In the next step, the hybridoma
supernatant was incubated undiluted and at several dilutions for
two hours at room temperature. After three additional washing steps
bound monoclonal antibody was detected with a horseradish
peroxidase conjugated polyclonal antibody against mouse
immunoglobulin. After 5 times of washing, the ELISA was finally
developed by addition of TMB-substrate solution
(Tetramethylbenzidine, Roche Mannheim). The colored precipitate was
measured after 15 min. at 405 nm using an ELISA-reader.
[0168] Supernatants from 10 clones exhibiting strong ELISA-signals
were selected for further analysis. In order to identify those
hybridoma clones, that produce monoclonal antibodies reactive with
native NKG2D-antigen on intact NK-cells and T-lymphocytes, the
following flowcytometric analysis was performed:
[0169] 1.times.10.sup.6 PBMC were incubated for 30 min. on ice with
50 .mu.l undiluted hybridoma supernatant and bound monoclonal
antibody was detected subsequently detected with fluorescein (FITC)
conjugated F(ab').sub.2 fragment of a rabbit anti-mouse Ig antibody
(Dako Hamburg, Code No. F0313) diluted 1:100 in PBS. In the next
step, the free valences of cell-bound FITC-conjugated antibody were
blocked through incubation of the cells for 30 minutes with 50
.mu.l mouse serum (Sigma immunochemicals, Deisenhofen, M-5905)
diluted 1:10. To distinguish between NK- and T-cells, labeled PBMC
were split at this point. One half was stained with a T-cell
specific tricolor conjugated anti-CD8 antibody (Caltac
Laboratories; Burlingame; USA, Code No. MHCD0306) diluted 1:100;
the other half was stained with an NK-cell specific phycoerythrin
(PE) conjugated anti-CD56 antibody (Becton Dickinson, Heidelberg,
Cat. No. 347747) diluted 1:25. Unlabeled anti-CD16 and anti-CD6
antibodies specifically staining NK-cells or T-lymphocytes,
respectively, were used as positive controls of the primary
labeling step; a murine monoclonal antibody with irrelevant
specificity instead of hybridoma supernatants reactive with
recombiant NKG2D served as negative control.
[0170] Cells were analyzed by flowcytometry on a FACS-scan (Becton
Dickinson, Heidelberg). FACS-staining and measuring of the
fluorescence intensity were performed as described in Current
Protocols in Immunology (Coligan, Kruisbeek, Margulies, Shevach and
Strober, Wiley-Interscience, 1992).
[0171] Two-color fluorescence analysis was carried out by applying
a positive gate for CD8.sup.+- and CD56.sup.+-cells, respectively,
thus allowing the detection of FITC-mediated fluorescence
separately on CD8.sup.+-T-lymphocytes and NK-cells. Compared with
the distinct staining of CD8.sup.+-T-lymphocytes and NK-cells with
the respective control antibodies, the supernatant of hybridoma
cell line 8R23 showed strong reactivity with both NK- and T-cells,
whereas two further supernatants were only weakly reactive with
both lymphocyte subsets.
[0172] Alternatively, monoclonal antibodies against human NKG2D
were generated by genetic immunization of mice. For this purpose,
two different fragments of human NKG2D from nucleotides (nt) 64 to
462 and from nt 123 to 462 corresponding to amino acid sequences
SEQ ID 3 and 4 were PCR-amplified from the cDNA-template shown in
FIG. 1, that encode extracellular NKG2D-segments flanked by
asparagine (N) and valine (V) or by tryptophan (W) and valine (V),
respectively. As PCR-primers the following oligonucleotides were
used:
[0173] NKG2D-short-f (5'-ATCAAGCTTGTGGATATGTTACAAAAATAACT-3') (SEQ
ID 80) and NKG2D-stop-r (5'-CGCGGTGGCGGCCGCTTACACAGTCCTTTGCATG-3')
(SEQ ID 82) for the amplification of the NKG2D-fragment: nt
123-462
[0174] as well as NKG2D-f (5'-ATCAAGCTTGAACCAAGAAGTTCAAATTCC-3')
(SEQ ID 81) and NKG2D-stop-r
(5'-CGCGGTGGCGGCCGCTTACACAGTCCTTTGCATG-3') (SEQ ID 82) for the
amplification of the NKG2D-fragment: nt 64-462.
[0175] Plasmids for genetic immunization were constructed by
cloning each of these PCR-products in-frame into the restriction
endonuclease sites Hind III and Not I of the vector VV1 (GENOVAC
AG, Germany) as shown in FIG. 4.
[0176] The resulting plasmids VV1-NKG2-D (nt 64-462) and VV1-NKG2-D
(nt 123-462) allowed the secretion of soluble extracellular NKG2-D
fragments tagged by a myc epitope at the N-terminus. The myc
epitope was utilized to confirm expression of the soluble NKG2-D
fragments. To this end the constructs were expressed by transient
transfection into BOSC-23 cells (Onishi (1996) Exp Hematol 24:
324), perforated by addition of Cytoperm/Cytofix (Becton
Dickinson); myc-tagged NKG2-D fragments were stained
intracellularly by FACScan analysis after reaction with a murine
anti-myc monoclonal antibody (9E10, ATCC, CRL-1729) followed by a
polyclonal phycoerythrin-labeled rabbit anti-mouse immunoglobulin
antibody.
[0177] Three 6 to 8 weeks old BALB/c mice were immunized six times
with VV1-NKG2-D (nt 64-462) and two mice were immunized three times
with VV1-NKG2-D (nt 64-462) followed by three immunizations with
VV1-NKG2-D (nt 123-462) using a Helios gene gun (Bio-Rad, Germany)
according to a published procedure (Kilpatrick (1998) Hybridoma 17:
569). One week after the last application of the immunization
plasmids each mouse was boosted by intradermal injection of 300
.mu.l of recombinant human NKG2-D protein (see Example 1)
concentrated 50 .mu.g/ml in phosphate buffered saline without
Mg.sup.2+ and Ca.sup.2+ ions at the DNA application sites.
[0178] Four days later, the mice were killed and their lymphocytes
were fused with SP2/0 mouse myeloma cells (American Tissue Type
Collection, USA) using polyethylene glycol (HybriMax;
Sigma-Aldrich, Germany), seeded at 100,000 cells per well in
96-well microtiter plates and grown in 200 .mu.l DMEM medium
supplemented with 10% fetal bovine serum and HAT additive for
hybridoma selection (Kilpatrick (1998) Hybridoma 17: 569).
[0179] Culture supernatants from densely grown wells were tested by
ELISA on immobilized recombinant NKG2D as described above.
Supernatants from 122 clones exhibiting positive ELISA-signals were
selected for further analysis. In order to identify those hybridoma
clones, that produce monoclonal antibodies reactive with native
NKGb-antigen on intact NK-cells and CD8.sup.+ T-lymphocytes, cells
were analysed by flowcytometry on a FACS-scan (Becton Dickinson,
Heidelberg).
[0180] Mononucleated cells from the peripheral blood (PBMC) of a
healthy donor were isolated by Ficoll-density gradient
centrifugation. In each well of a microtiter plate 200.000 PBMC
were incubated with undiluted hybridoma supernatant. After 30
minutes of incubation on ice cells were washed twice with PBS and
subsequently stained with fluorescein (FITC)-conjugated
F(ab').sub.2 fragment of a goat anti-mouse IgG and IgM antibody
(Jackson ImmunoResearch Inc. West Grove, USA, Code 115-096-068;
1:100) for 30 minutes on ice. The cells were washed twice with PBS
and subsequently stained with two different antibody labeling
mixtures. For staining of CD8.sup.+ T cells, 100.000 PBMC were
further incubated for 30 minutes with a phycoerythrin (PE)
conjugated CD16 antibody (Becton Dickinson, Heidelberg, Code No.
347617) and a tricolor conjugated CD8 antibody (Caltac
Laboratories, Burlingame, USA, Code No. MHCD0806). For staining of
NK-cells, the other half of the PBMC was further incubated for 30
minutes with a phycoerythrin (PE) conjugated CD56 antibody (Becton
Dickinson, Heidelberg, Code No. 347747) and a tricolor conjugated
CD3 antibody (Caltac Laboratories, Burlingame, USA, Code No.
MHCD0306.). In order to avoid cross-reactions between the different
antibodies within the labeling mixtures mouse serum (Sigma Aldrich,
St. Louis, USA, Cat. No. 054H-8958) was added at a final dilution
of 1:10.
[0181] Triple color fluorescence analysis was carried out by
applying a positive gate for CD8.sup.+ (Tricolor) and a negative
gate for CD16.sup.+ (PE) cells, thus allowing the detection of
FITC-mediated fluorescence exclusively attributed to CD8.sup.+
T-lymphocytes (phenotype: CD8.sup.+, CD16.sup.-) without any
contaminating signals from CD8.sup.+ NK-cells. Similarly, triple
color fluorescence analysis as carried out by applying a positive
gate for CD56.sup.+, (PE) and a negative gate for CD3.sup.+-cells
(tricolor) thus allowing the detection of FITC-mediated
fluorescence exclusively attributed to NK-cells (phenotype:
CD56.sup.+, CD3.sup.-) without any contaminating signals from
CD56.sup.+-T lymphocytes. As shown in FIG. 5, the supernatants of
the hybridomas designated 11B2, 8G7 and 6E5 contained monoclonal
antibodies reactive with native NKG2D on the surface of both human
CD8.sup.+ T-lymphocytes and NK-cells. Staining with supernatant of
the hybridoma 10H9 is shown as a representative example of many
monoclonal antibodies reactive with immobilized recombinant NKG2D,
that were, however, not capable of binding the native
NKG2D-receptor complex on intact cells. FACS staining and measuring
of the fluorescence intensity were performed as described in
Current Protocols in Immunology (Coligan, Kruisbeek, Margulies,
Shevach and Strober, Wiley-Interscience, 1992).
[0182] The hybridomas producing antibodies reacting with NKG2-D on
CD56.sup.+ NK- and CD8.sup.+ T cells were subcloned once by limited
dilution on 96-well microtiter plates. Positive subclones were
identified by flowcytometry on NKG2D-positive NKL-cells (Bauer
(1999) Science 285: 727) incubated with supernatants harvested from
wells showing cell growth. Cell-bound monoclonal antibody was
detected with the fluorescein (FITC) conjugated
F(ab').sub.2-fragment of a rabbit anti-mouse Ig antibody (Dako,
Hamburg, Code No. F0313). The subclones 11B2D10, 8G7C10 and 6E5A7
were further used for the construction of NKG2D-directed bispecific
antibodies (see Example 3).
EXAMPLE 3
[0183] Construction of Bispecific Single-Chain Antibodies
Anti-NKG2D.times.Anti-EpCAM
[0184] The bispecific antibodies were constructed as depicted in
FIG. 2. The variable regions V.sub.L and V.sub.H of those
antibodies binding to native NKG2D on intact cells were cloned from
total RNA of the corresponding hybridoma cell lines as described by
Orlandi (1989) Proc. Natl. Acad. Sci. USA 86: 3833, except that the
PCR-fragments of variable regions amplified from hybriomas 11B2D16
(SEQ ID 7-16), 8G7C10 (SEQ ID 27-36), 6E5A7 (SEQ ID 37-46) and
6H7E7 (SEQ ID 17-26) were directly the TA-cloning vector GEM-T Easy
(Promega, Cat. No. A1360). Subsequently, cloned VL- and VH-regions
served as templates for a two-step fusions-PCR resulting in the
corresponding scFv-fragments with the domain arrangement VL/VH. The
VL-specific primer pair used for this purpose consists of
oligonucleotides 5'V.sub.LB5RRV (5'AGG TGT ACA CTC CGA TAT CCA GCT
GAC CCA GTC TCC A 3' (SEQ ID 83)) and 3.dbd.VLGS15 (5'GGA GCC GCC
GCC GCC AGA ACC ACC ACC ACC TTT GAT CTC GAG CTT GGT CCC3' (SEQ ID
84)), the VH-primer pair of oligonucleotides 5'V.sub.HGS15 (5'GGC
GGC GGC GGC TCC GGT GGT GGT GGT TCT CAG GT(GC) (AC)A(AG) CTG CAG
(GC)AG TC(AT) GG 3' (SEQ ID 85)) and 3'VHBspEI (5'AAT CCG GAG GAG
ACG GTG ACC GTG GTC CCT TGG CCC CAG 3' (SEQ ID 86)). In the first
PCR step VH- and VL-amplification products were obtained with the
following PCR-programm: denaturation at 94.degree. C. for 5 min,
annealing at 37.degree. C. for 2 min, elongation at 72.degree. C.
for 1 min for the first cycle; denaturation at 94.degree. C. for 1
min, annealing at 37.degree. C. for 2 min, elongation at 72.degree.
C. for 1 min for 6 cycles; denaturation at 94.degree. C. for 1 min,
annealing at 55.degree. C. for 1 min, elongation at 72.degree. C.
for 45 sec and 18 cycles; termial extension at 72.degree. C. for 2
min. For the second step of the fusion PCR VH- and VL-PCR fragments
were purified from agarose gel, mixed with oligonucleotide primers
5'VLB5RRV and 3'VHBspEI, and subjected to the following
PCR-programm: denaturation at 94.degree. C. for 5 min once;
denaturation at 94.degree. C. for 1 min, annealing at 55.degree. C.
for 1 min, elongation at 72.degree. C. for 1,5 min and 8 cycles;
terminal extension at 72.degree. C. for 2 min. VL/VH-fusion
products encoding anti-NKG2D scFv-fragments were purified from
agarose gele, and digested with the restriction enzymes
BsrGI/BspEI. The mammalian expression vector pEF-DHFR (Mack (1995)
Proc Natl Acad Sci USA 92: 7021) containing an EcoRI/SalI-cloned
DNA-fragment described in WO0003016, FIG. 10 was also digested with
the restriction enzymes BsrGI/BspEI releasing a 750 bp-fragment;
the remaining vector-fragment was gele purified and used for
cloning of the anti-NKG2D scFv-fragments.
[0185] Thus, the resulting derivatives of mammalian expression
vector pEF-DHFR contain EcoRI/SalI-DNA inserts encoding bispecific
single-chain antibodies as described by Mack (1995) Proc Natl Acad
Sci USA 92: 7021, that are directed against NKG2D and EpCAM. EpCAM
is expressed by many epithelial tumors and already used as target
antigen for the adjuvant treatment of resected colorectal cancer
with a murine monoclonal antibody.
[0186] The expression plasmids for anti-NKG2D.times.anti-EpCAM
bispecific single-chain antibodies (SEQ ID 47-49) were transfected
into DHFR-deficient CHO-cells by electroporation; selection for
stable transfectants, gene amplification and protein production
were performed as described (Mack (1995) Proc Natl Acad Sci USA 92:
7021). Bispecific antibody was purified from culture supernatant
via the C-terminal histidine tag by using Ni-NTA-column as
described (Mack (1995) Proc Natl Acad Sci. USA 92: 7021, see also
FIG. 3).
EXAMPLE 4
[0187] Antibodies Directed to the Extracellular Domain of
DAP-10
[0188] Antibodies reactive with the extracellular domain of the
NKG2D-receptor complex can be also be obtained with the following
protocol:
[0189] 6 to 8 weeks old BALBlc mice may be immunized with a peptide
corresponding to the complete extracellular domain of human DAP-10
comprising 30 amino acids (SEQ ID 5,
QTTPGERSSLPAFYPGTSGSCSGCGSLSLP) or a part thereof (Wu (1999)
Science 285: 730), conjugated with a carrier protein, respectively.
For example, a peptide comprising the 21 N-terminal amino acids of
the extracellular domain of DAP10 (SEQ ID 6, QTTPGERSSLPAFYPGTSGSC)
may be coupled to maleinimide activated KLH in a directed manner
via the mercapto-group of its C-terminal cystein. The conjugate may
be dissolved in 0,9% NaCl at a concentration of 100 .mu.g/ml, the
solution subsequently emulsified 1:2 with complete Freund's
adjuvants and 50 .mu.l per mouse infected intraperitonially. Mice
may receive booster immunizations resembling the primary
immunization after 4, 8 and 12 weeks, except that complete Freund's
adjuvants can be replaced by incomplete Freund's adjuvants. Ten
days after the first booster immunization, blood samples may be
taken and antibody serum titer tested by ELISA on immobilized BSA
conjugated with the 21-mer DAP-10 peptide as described above for
KLH.
[0190] Three days after the second boost, spleen cells from mice
with positive serum titer may be fused with P3.times.63Ag8.653
cells (ATCC CRL-1580) to generate hybridoma cell lines following
standard protocols as described in Current Protocols in Immunology
(Coligan, Kruisbeek, Margulies, Shevach and Strober,
Wiley-Interscience, 1992). After PEG-fusion, cells may be seeded at
100.000 cells per well in microtiterplates and grown in 200 .mu.l
RPMI 1640 medium supplemented with 10% fetal bovine serum, 300
units/ml recombinant human interleukin 6 and HAT-additive for
selection. Culture supernatants from densely grown wells may be
tested for reactivity with DAP10-peptide by the following
ELISA:
[0191] The wells of a 96 U-bottom plate (Nunc, maxisorb) are coated
overnight at 4.degree. C. with peptide-BSA-conjugate at a
concentration of 5 .mu.g/ml. Coated wells are washed three times
with washing buffer (0.1M NaCl, 0.05M Na.sub.2HPO.sub.4 pH 7.3,
0.05% Tween 20, 0.05% NaN.sub.3) and subsequently blocked through
incubation for one hour at room temperature with 200 .mu.l/well of
2% skimmed milk powder suspended in washing buffer. In the next
step, hybridoma supernatant is incubated undiluted and at several
dilutions for two hours at room temperature. After three additional
washing steps bound monoclonal antibody can be detected with a
horseradish peroxidase conjugated polyclonal antibody against mouse
immunoglobulin. After 5 times of washing, the ELISA can be finally
developed by addition of TMB-substrate solution
(Tetramethylbenzidine, Roche). The colored precipitate is measured
after 15 min. at 405 nm using an ELISA-reader.
[0192] In order to identify those peptide-reactive hybridoma
clones, that produce monoclonal antibodies capable of binding to
DAP-10 within the NKG2D-receptor complex on intact NK-cells and
CD8.sup.+ T-lymphocytes, the triple color fluorescence analysis on
PBMC may be performed that is described in Example 2.
[0193] The variable regions of monoclonal antibodies staining
intact NK-cells and CD8.sup.+ T-lymphocytes may be cloned from the
corresponding hybridoma cell lines and used for construction of
bispecific single-chain antibodies as described in Example 3.
EXAMPLE 5
[0194] Enhanced Priming of Naive CD8.sup.+ T Cells by
NKG2D-Directed Antibodies
[0195] For in vitro priming experiments naive human CD8.sup.+ T
cells were isolated as follows:
[0196] Mononuclear cells (PBMC) were prepared by Ficoll density
centrifugation from 500 ml peripheral blood obtained from a healthy
donor. CD8.sup.+-T cells were isolated by negative selection using
a commercially available cell separation kit (R&D Systems,
HCD8C-1000). The CD8.sup.+-T cell column was loaded with
2.times.10.sup.8 PBMC, which had been preincubated with the
manufacture's antibody cocktail supplemented with 1 .mu.g of a
monoclonal anti-CD11b antibody (Coulter 0190) per column. Since
primed non-proliferating cytotoxic CD8.sup.+ T cells share the
CD45RA.sup.+/RO.sup.- phenotype with naive CD8.sup.+ T lymphocytes,
CD11b was introduced as additional cell purification marker in
order to get rid of the former T cell subset. Thus, only
CD11b.sup.-/CD8.sup.+ T cells entered the purification procedure
based on CD45-isoforms finally resulting in naive CD8.sup.+-T
lymphocytes, that like naive CD4.sup.+-T cells carry the
CD45RA.sup.+/RO.sup.- phenotype. Successful purification of
CD8.sup.+-T cells was controlled by flowcytometry after single
staining with an anti-CD8 antibody. Absence of CD11b.sup.+-cells
from CD8.sup.+-T cell preparations was confirmed by single staining
with an anti-CD28 antibody, since CD11b-positive CD8.sup.+-T cells
are always CD28-negative and vice versa.
[0197] CD45RO.sup.+-cells were removed from purified CD8.sup.+-T
cells through incubation with a murine monoclonal anti-CD45RO
antibody (PharMingen, UCHL-1, 31301) followed by magnetic beads
conjugated with a polyclonal sheep anti-mouse Ig antibody (Dynal,
110.01). The purity of the remaining naive CD8.sup.+-T cells proved
to be >95% as determined by flowcytometry after double staining
with anti-CD45RA/anti-CD45RO. The average yield of naive CD8.sup.+
T cells per 500 ml peripheral blood was 5.times.10.sup.6 (CD8).
[0198] The in vitro priming experiment with naive CD8.sup.+ T cells
was carried out as follows:
[0199] 25.000 EpCAM-transfected CHO-cells per well were incubated
in a 96-well flat-bottom culture plate for 2 hours, that had been
coated overnight with a polyclonal rabbit anti-mouse IgGl antibody
(Dako, Z0013) diluted 1:1000 in PBS. After the cells had adhered to
the plastic, they were irradiated with 14.000 rad. Subsequently,
50.000 purified naive CD8.sup.+ T cells per well were added in RPMI
1640 medium supplemented with 10% human AB serum, 100 U/ml
penicillin, 100 mg/ml streptomycin, 2 mM glutamin, 1 mM sodium
pyruvat, 10 mM HEPES-buffer, 1.times. non-essential amino acids
(Gibco) and 50 .mu.M .beta.-mercaptoethanol. The EpCAM-specific
B7-1/4-7 single-chain construct described in WO9925818 (Example 7)
was added at 500 ng/mI together with 1 .mu.g/ml of a murine IgG1
isotype control (Sigma, M-7894) and either 250 ng/ml, 50 ng/ml or
no bispecific single-chain antibody (bsc) EpCAM.times.CD3 (Mack
(1995) Proc. Natl. Acad. Sci. U.S.A. 92: 7021). 500 ng/mI of the
B7-1/4-7 single-chain construct was the maximum concentration that
did not yet by itself affect CD45-isoform expression on CD8.sup.+-T
cells. In a parallel experiment, the same concentrations and
combinations of bsc EPCAM.times.CD3 and B7-1/4-7 single-chain
construct were used except that the IgG1 isotype control was
replaced by diluted hybridoma supernatant kindly provided by Dr.
Moretta, Genova, Italy containing the murine NKG2D-specific IgG1
antibody BAT221 at a final concentration of 1 .mu.g/ml.
Alternatively the NKG2D-specific monoclonal antibody can be
exchanged for a bispecific antibody binding to NKG2D and EpCAM like
SEQ ID 47-49 and 72-79. In contrast to the monoclonal antibody no
bispecific antibody is immobilized on the solid support.
[0200] All experiments were carried out with triplicates of
identical wells. Furthermore, a set of two identical 96-well plates
was prepared in order to make sure, that enough cells were
available for flowcytometry on days 3 and 6. On day 3, supernatant
was harvested from one 96-well plate and TNF-.alpha. concentration
determined by using a commercially available ELISA-kit (PharMingen,
2600KK). The cells were also harvested and subjected to
flowcytometric analysis of CD45-isoform expression. Moreover, half
of the supernatant was removed from each well of the second 96-well
plate and replaced by fresh medium adjusted to the corresponding
concentrations of B7-1/4-7 single-chain construct, bsc
EpCAM.times.CD3, BAT221 and/or isotype control. On day 6, the cells
of this 96-well plate were harvested and their CD45-isoform
expression pattern was analyzed by flowcytometry. In general, cells
and supernatants from three identical wells (triplicates) were
pooled for flowcytometry and cytokine analysis, respectively.
[0201] Flowcytometry was performed on a FACScan (Becton Dickinson).
Flowcytometric analysis of CD45-isoform expression was carried out
by double staining of 1.times.10.sup.5 cells with a PE-conjugated
monoclonal anti-CD45RA antibody (Coulter, 2H4, 6603904) and a
FITC-conjugated monoclonal anti-CD45RO antibody (DAKO, UCHL-1, F
0800) for 30 minutes on ice. Flowcytometric monitoring of T cell
purification was equally carried out by single stainings with a
Tricolor-conjugated monoclonal anti-CD8 antibody (Medac, MHC0806)
and a FITC-conjugated monoclonal anti-CD28 antibody (Medac,
MHCD2801).
[0202] In these priming experiments, the primary signal was
mediated by the bispecific single-chain antibody (bscAb)
EpCAM.times.CD3 thus imitating specific antigen recognition through
the T-cell receptor (TCR); the second or costimulatory signal was
mediated by the EpCAM-specific B7-1/4-7 single-chain construct
through engagement of CD28 on the T-cells. Thus the effect of an
NKG2D-directed antibody on the priming of naive CD8.sup.+ T cells
could be determined, that were activated through the TCR-like and
the costimulatory signal. Non-human stimulator cells armed with
EpCAM-specific constructs were used in order to avoid background
signals, that may arise with human stimulator cells incidently
expressing costimulatory receptors. The kinetics of T cell priming
was monitored by flowcytometry on days 3 and 6 simultaneously
measuring the expression of CD45RA and CD45RO. As shown in FIG. 6,
almost the entire population of naive T cells changed the
CD45-phenotype to that of primed T cells, i.e.
CD45RA.sup.-/RO.sup.+, within 6 days in the presence of B7-1/4-7
single-chain construct (500 ng/ml) and bscAb EpCAM.times.CD3 (250
ng/ml). Accordingly, an intermediate state could be observed on day
3. Surprisingly, the additional presence of the NKG2D-directed
antibody further accelerated proliferation and priming of naive
CD8.sup.+ T cells. This could be concluded from the higher
percentage of CD8.sup.+ T-cells located on day 3 in the lower right
quadrant in FIG. 6B, if the cells had received the additional
NKG2D-signal compared to a smaller percentage when the naive
T-cells were stimulated through the TCR-like and costimulatory
signal alone. Since TNF-.alpha. is typically produced by primed
CD8.sup.+-T cells but not by their naive counterparts, the effect
of enhanced T-cell priming could be confirmed by higher
concentrations of TNF-.alpha. measured on day 3 in the supernatant
of CD8.sup.+ T-cells receiving the NKG2D-signal compared to those
primed in the absence of the NKG2D-directed antibody (FIG. 7).
[0203] Even the flowcytometric results on day 6 (FIGS. 6C and E),
when the priming process was almost completed, showed the
NKG2D-mediated support of T cell priming: The loss of
CD45RA-expression within 6 days proved to be more profound in the
presence than in the absence of an NKG2D-mediated signal as
measured by the higher percentage of CD8.sup.+ T cells located
within the lower right field in FIG. 6C.
EXAMPLE 6
[0204] NKG2D-Directed Antibodies Enhance the Cytotoxicity of
CD8.sup.+ T-Cells and NK-Cells Triggered through Engagement of the
TCR- or the Fc.gamma.RIII-Complex, Respectively
[0205] In order to test the recruitment of cytotoxic lymphocytes
i.e. CD8.sup.+ T cells and NK cells by NKG2D-directed antibodies,
we performed .sup.51Cr-release assays using the murine
Fc.gamma.R-positive P815 cell line as target and either a Melan-A
specific human CD8.sup.+ T cell clone (Melan-A cells) or NKL cells
(Bauer (1999) Science 285: 727) as effectors. The .sup.51Cr-release
assay measuring cellular cytotoxicity was carried out as described
by Mack (1995) Proc Natl Acad Sci USA 92: 7021 with minor
modifications. 10.000 .sup.51Cr-labeled P815-cells were either
mixed with 50.000 Melan-A cells or with 200.000 NKL cells per well
of a round-bottomed microtiter plate. NKL cells were incubated for
4 h in the presence of 5.mu.g/ml CD16 antibody (3G8) and/or diluted
hybridoma supernatant containing the murine NKG2D-specific
monoclonal antibody BAT221. Melan A cells were incubated for 4 h in
the presence of 0,2 .mu.g/ml CD3 antibody (OKT3) and/or diluted
BAT221-supernatant. Maximum .sup.51Cr-release was determined by
lysis of target cells with Maly buffer (2% SDS/0,37% EDTA/0,53%
Na.sub.2CO.sub.3). The spontaneous .sup.51Cr-release was determined
with target cells incubated in the absence of effector cells and
antibodies. Target cells incubated with effector cells in the
absence of antibodies served as negative control. Specific lysis
was calculated as [(cpm, experimental release)-(cpm, spontaneous
release)]/[(cpm, maximal release)-(cpm, spontaneous release)]. The
cytotoxicity assay was carried out with triplicate samples. As
shown in FIG. 8, BAT221 did not induce any substantial target cell
lysis by itself in contrast to published NKG2D-antibodies (Bauer
(1999) Science 285: 727). As expected the CD16- and the
CD3-antibody induced redirected target cell lysis with NKL-cells
and Melan A-cells, respectively. However, although not cytotoxic by
itself, BAT221 surprisingly enhanced target cell cytotoxicity
triggered by engagement of the TCR-complex on Melan A-cells and of
the Fc.gamma.RIII-complex on NKL-cells. Alternatively, P815-cells
can be replaced e.g. by EpCAM-positive Kato-cells and the
NKG2D-specific monoclonal antibody exchanged for a bispecific
antibody binding to NKG2D and EPCAM like SEQ ID 47-49 and 72-79.
The TCR-complex on CD8.sup.+ T-cells may be engaged by a bispecific
antibody binding to CD3 and to a surface antigen on the target
cells or by specific TCR-recognition of processed MHC I-complexed
target cell antigen. The Fc.gamma.RIII-complex on NK-cells may be
engaged by a bispecific antibody binding to CD16 and to a surface
antigen on the target cells or by a target cell specific monoclonal
antibody like e.g. a human EpCAM antibody bound to Fc.gamma.RIII
via its Fc part.
EXAMPLE 7
[0206] Bispecific Single-Chain Antibodies with an NKG2D-Binding
Site Located C-Terminally of the Target Binding Site
[0207] Five Balb/c mice were genetically immunized with human NKG2D
as described in Example 2. In order to identify mice with a serum
antibody reactivity on the surface of human lymphocytes resembling
the expression pattern of the NKG2D-receptor complex, a triple
fluorescence analysis on PBMC as described in Example 2 was carried
out with mouse serum diluted 1:10, 1:20 and 1:40. As shown in FIG.
9, only one mouse serum (No. 4) exhibited strong staining of both
CD8.sup.+ T-lymphocytes and NK-cells. The spleen cells of this
mouse were used as an immunoglobulin repertoire for the
construction of a combinatorial antibody library as described in
WO9925818 (Example 6). The cloned antibody repertoire was displayed
on filamentous phage as an N2-VH-VL-fusion protein, imitating the
C-termial position of the corresponding antigen binding site within
a bispecific single-chain antibody. Selection of NKG2D-reactive
scFv-fragments was carried out through two rounds of library
panning on immobilized recombinant NKG2D-protein as described in
WO9925818 for the 17-1A- or EpCAM-antigen followed by three rounds
of panning on NKG2D-positive NKL-cells. Cell panning was carried
out in PBS/10%FCS by resuspending 2-5.times.10.sup.6 NKL-cells in
500 .mu.l phage suspension followed by 45 minutes of moderate
shaking at 4.degree. C. Then cells plus bound phage particles were
centrifuged in a desk centrifuge at 2500 rpm for 2 minutes. Then
the resulting pellet was washed twice by resuspension in 1 ml of
PBS/10%FCS followed by recentrifugation 4-third round of panning).
During the fourth round of panning 3 washing steps were applied as
well as 5 washing steps during the fifth round of panning.
Specifically bound phage particles were eluted from the NKL-cells
by resuspension and incubation in 500 .mu.l HCl-Glycin for 10
minutes followed by neutralization with 30 .mu.l 2M Tris-base (pH
12). The eluant were used for infection of a new uninfected E. coli
XL1 Blue culture. After five rounds of phage-production and
subsequent selection for antigen-binding scFv-displaying phages,
plasmid DNAs from E. coli cultures were isolated corresponding to
the fourth and fifth round of panning. For the production of
soluble scFv-antibody fragments that carry the N2-domain at their
N-terminus, the DNA fragment encoding the CT-domain of the
geneIII-product was excised with SpeI/NotI and replaced by the
tetramerization domain of human p53 (Rheinnecker (1996) J Immunol.
157: 2989) flanked by an N-terminal Ig-hinge-region and a
C-terminal Flag-epitope (FIG. 10, SEQ ID 50 and 51). After
ligation, the resulting pool of plasmid DNA was transformed into
100 .mu.l of heat shock competent E. coli XL1 Blue cells and plated
on Carbenicilline LB-agar. Single colonies were check by
screening-PCR for integrity of the cloned VH- and VL-regions and
those with intact variable regions subjected to periplasmatic
expression of soluble antibody fragments as described in WO9925818
(Example 6). The periplasma preparations were tested by ELISA on
immobilized recombinant NKG2D and specifically binding
N2-scFv-p53-fusion proteins detected with the peroxidase-conjugated
anti-Flag M2 antibody (Sigma, A-8592). As shown in FIG. 11 many
NKG2D-reactive clones from the fourth and fifth round of panning
could be identified. The scFv-encoding fragments of the positive
clones were excised with BspEI from the phage display vector and
subcloned into the plasmid vector BS-CTI (see WO 00-06605, FIG. 2)
prepared by digestion with BspEI and XmaI followed by
dephosphorylation with calf intestinal phophatase. The correct
orientation of the scFv-fragments was checked by analytic digestion
with the restriction enzymes BspEI and SpeI. By insertion into
BS-CTI the scFv-encoding fragments were fused in-frame to a
His.sub.6-tag (SEQ ID 52-71). In the next step, the scFv-fragments
were excised with BspEI and SalI from BS-CTI and subcloned
BspEI/SalI into the mammalian expression vector PEF-DHFR that
contained an EpCAM-specific, CD3-directed bispecific single-chain
antibody as described by Mack (1995) Proc Natl Acad Sci USA 92:
7021 except that the scFv-fragment of the EpCAM-specific monoclonal
antibody M79 had been replaced by that of the monoclonal antibody
3B10, that binds to EpCAM with high affinity (=bsc 3B10.times.CD3).
Thus, the CD3-specific scFv-fragment was replaced by NKG2D-reactive
scFv-fragments resulting in EpCAM-specific, NKG2D-directed
bispecific single-chain antibodies (SEQ ID 72-79).
[0208] CHO/dhfr- cells were chosen for the transient expression of
the antibody-like molecules. The transfection of the cells was
performed with the TransFast transfection reagent (Promega,
Heidelberg) according to the manufacture's protocol. Briefly,
2.5.times.10.sup.5 cells were seeded per well in six-well plates 20
hrs prior to transfection. The transfection mix was prepared by
adding 6 .mu.g of plasmid DNA harboring the antibody sequences or
the .beta.-galactosidase gene to 1 ml MEM alpha media without
supplements. After mixing 30 .mu.l of TransFast reagent were added.
The mix was vortexed and incubated for 15 minutes at room
temperature. Then, the media was removed from the cells and
replaced by the transfection mix. After 1 hour incubation period at
37.degree. C. the transfection mix was aspirated and fresh complete
MEM alpha was added to the cells. Protein production was analyzed 4
to 5 days post transfection by FACS analysis. The supernatants were
harvested after 4 to 5 days. To remove cell debris the supernatants
were centrifuged. The function of the antibodies was analyzed in
binding studies of the anti-EpCAM specific part on Kato III cells.
Per sample, 4.times.10.sup.5 cells were incubated in 75 .mu.l
transfected cell supernatant diluted with 25 .mu.l FACS buffer (1%
heat-inactivated FBS, 0.05% Na3N in PBS). The samples were
incubated for 30 minutes at 4.degree. C. After washing the cells
twice with 200 .mu.l FACS buffer the cells were incubated with 2
.mu.g/ml anti-Penta.His antibody (QIAGEN, Netherlands) for 30
minutes at 4.degree. C. Subsequently, the cells were washed again
and incubated for 30 minutes with a sheep anti-mouse FITC conjugate
(SIGMA, Deisenhofen. Binding was detected with a FACS Calibur
(Becton-Dickinson) (FIG. 12). Supernatants of CHO-cells transiently
transfected with bsc 3B10.times.P4-3 and bsc 3B10.times.P5-2 showed
positive ELISA-signals on immobilized recombinant NKG2D-antigen
(FIG. 13).
[0209] As an alternative to NKG2D, DAP10-peptide-conjugates as
described in Example 4 may be used for immunizing mice, whose
spleen cells may be the source of an immunoglobulin repertoire for
the construction of a combinatorial antibody library like that
described in this Example. Thus, DAP10 reactive antibody binding
sites recognizing the NKG2D-receptor complex on CD8.sup.+
T-lymphocytes and NK-cells even when located C-terminally of the
target binding site within a bispecific single-chain antibody may
be selected through library panning on immobilized
peptide-conjugate and/or cells expressing the NKG2D-receptor
complex.
EXAMPLE 8
[0210] Recruitment of CD8.sup.+ T and NK Effector Cells by
Bispecific Single-Chain Antibodies with an NKG2D-Binding Site
Located C-Terminally of the Target Binding Site
[0211] In order to test the recruitment of cytotoxic lymphocytes
i.e. CD8.sup.+ T cells and NK cells by NKG2D-directed bispecific
antibodies, we performed .sup.51Cr-release assays using the gastric
cancer cell line Kato as target and either a Melan-A specific human
CD8.sup.+ T cell clone (Melan-A cells), NKL cells (Bauer (1999)
Science 285: 727) or unstimulated PBMC from a healthy donor as
effectors.
[0212] The .sup.51Cr-release assay measuring cellular cytotoxicity
redirected against EpCAM-positiv Kato-cells was carried out as
described by, Mack (1995) Proc Natl Acad Sci USA 92: 7021 with
minor modifications. 10.000 .sup.51Cr-labeled Kato cells were
either mixed with 50.000 Melan-A cells or with 200.000 NKL cells or
PBMC per well of a round-bottomed microtiter plate and incubated
for 4 h (Melan A- and NKL-cells) or 18 h (PBMC) in the presence of
culture supernatant from CHO cells diluted 1:2 that had been
transfected with different EpCAM-specific, NKG2D-directed
bispecific single-chain antibodies (3B10.times.P4-3,
3B10.times.P4-14, 3B10.times.P5-2 and 3B10.times.P5-23) described
in Example 8. Maximum .sup.51Cr-release was determined by lysis of
target cells with Maly buffer (2% SDS/0,37% EDTA/0,53%
Na.sub.2CO.sub.3). The spontaneous .sup.51Cr-release was determined
with target cells incubated in the absence of effector cells and
bispecific antibody. Target cells incubated with effector cells in
the absence of antibodies served as negative control. Specific
lysis was calculated as [(cpm, experimental release)-(cpm,
spontaneous release)]/[(cpm, maximal release)-(cpm, spontaneous
release)]. The cytotoxicity assay was carried out with triplicate
samples. As shown in FIG. 14, the supernatant of CHO-cells
transiently transfected with the NKG2D-directed bispecific
single-chain antibody 3B10.times.P4-3 induced weak but reproducible
and titratible cytolyis of EpCAM-positive Kato-cells with both
Melan-A- and NKL-cells in the 4h-.sup.51Cr release test. Moreover,
the supernatants of CHO-cells transiently transfected with the
NKG2D-directed bispecific single-chain antibodies 3B10.times.P4-3,
3B10.times.P4-14, 3B10.times.P5-2 and 3B10.times.P5-23,
respectively induced substantial target cell lysis with PBMC in the
18 h .sup.51Cr-release assay (FIG. 15).
Sequence CWU 1
1
92 1 429 DNA Homo sapiens 1 ttattcaacc aagaagttca aattcccttg
accgaaagtt actgtggccc atgtcctaaa 60 aactggatat gttacaaaaa
taactgctac caattttttg atgagagtaa aaactggtat 120 gagagccagg
cttcttgtat gtctcaaaat gccagccttc tgaaagtata cagcaaagag 180
gaccaggatt tacttaaact ggtgaagtca tatcattgga tgggactagt acacattcca
240 acaaatggat cttggcagtg ggaagatggc tccattctct cacccaacct
actaacaata 300 attgaaatgc agaagggaga ctgtgcactc tatgcctcga
gctttaaagg ctatatagaa 360 aactgttcaa ctccaaatac atacatctgc
atgcaaagga ctgtgtccgg gcatcatcac 420 catcatcat 429 2 143 PRT Homo
sapiens 2 Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr
Cys Gly 1 5 10 15 Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn
Cys Tyr Gln Phe 20 25 30 Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser
Gln Ala Ser Cys Met Ser 35 40 45 Gln Asn Ala Ser Leu Leu Lys Val
Tyr Ser Lys Glu Asp Gln Asp Leu 50 55 60 Leu Lys Leu Val Lys Ser
Tyr His Trp Met Gly Leu Val His Ile Pro 65 70 75 80 Thr Asn Gly Ser
Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn 85 90 95 Leu Leu
Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala 100 105 110
Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr 115
120 125 Ile Cys Met Gln Arg Thr Val Ser Gly His His His His His His
130 135 140 3 133 PRT Homo sapiens 3 Asn Gln Glu Val Gln Ile Pro
Leu Thr Glu Ser Tyr Cys Gly Pro Cys 1 5 10 15 Pro Lys Asn Trp Ile
Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp 20 25 30 Glu Ser Lys
Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn 35 40 45 Ala
Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu Lys 50 55
60 Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His Ile Pro Thr Asn
65 70 75 80 Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn
Leu Leu 85 90 95 Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu
Tyr Ala Ser Ser 100 105 110 Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr
Pro Asn Thr Tyr Ile Cys 115 120 125 Met Gln Arg Thr Val 130 4 114
PRT Homo sapiens 4 Trp Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe
Asp Glu Ser Lys 1 5 10 15 Asn Trp Tyr Glu Ser Gln Ala Ser Cys Met
Ser Gln Asn Ala Ser Leu 20 25 30 Leu Lys Val Tyr Ser Lys Glu Asp
Gln Asp Leu Leu Lys Leu Val Lys 35 40 45 Ser Tyr His Trp Met Gly
Leu Val His Ile Pro Thr Asn Gly Ser Trp 50 55 60 Gln Trp Glu Asp
Gly Ser Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile 65 70 75 80 Glu Met
Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly 85 90 95
Tyr Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg 100
105 110 Thr Val 5 30 PRT Homo sapiens 5 Gln Thr Thr Pro Gly Glu Arg
Ser Ser Leu Pro Ala Phe Tyr Pro Gly 1 5 10 15 Thr Ser Gly Ser Cys
Ser Gly Cys Gly Ser Leu Ser Leu Pro 20 25 30 6 21 PRT Homo sapiens
6 Gln Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr Pro Gly 1
5 10 15 Thr Ser Gly Ser Cys 20 7 321 DNA Artificial Sequence
Description of Artificial Sequence Synthetic Anti-NKG2D hybridoma
11B2D10 variable light chain 7 gacattcagc tgacccagtc tccagcctcc
ctatctgcat ctgtgggaga aactgtcacc 60 atcacatgtc gagcaagtgg
gaatattcac aattatttag cttggtatca gcagaaacag 120 ggaaaatctc
ctcaggtcct ggtctataat gcaaaaacct tagcagatgg tgtgccatca 180
aggttcagtg gcagtggatc aggaacacaa tattctctca agatcaacag cctgcagcct
240 gaagattttg ggagttatta ctgtcaacat ttttggagta ctacgtggac
gttcggtgga 300 gggaccaagc tggagatcaa a 321 8 107 PRT Artificial
Sequence Description of Artificial Sequence Synthetic Anti-NKG2D
hybridoma 11B2D10 variable light chain 8 Asp Ile Gln Leu Thr Gln
Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr
Ile Thr Cys Arg Ala Ser Gly Asn Ile His Asn Tyr 20 25 30 Leu Ala
Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Val Leu Val 35 40 45
Tyr Asn Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln
Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Ser
Thr Thr Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 9 11 PRT Artificial Sequence Description of Artificial
Sequence Synthetic Anti-NKG2D hybridoma 11B2D10 variable light
chain CDR1 9 Arg Ala Ser Gly Asn Ile His Asn Tyr Leu Ala 1 5 10 10
7 PRT Artificial Sequence Description of Artificial Sequence
Synthetic Anti-NKG2D hybridoma 11B2D10 variable light chain CDR2 10
Asn Ala Lys Thr Leu Ala Asp 1 5 11 9 PRT Artificial Sequence
Description of Artificial Sequence Synthetic Anti-NKG2D hybridoma
11B2D10 variable light chain CDR3 11 Gln His Phe Trp Ser Thr Thr
Trp Thr 1 5 12 351 DNA Artificial Sequence Description of
Artificial Sequence Synthetic Anti-NKG2D hybridoma 11B2D10 variable
heavy chain 12 caggtccaac tgcagcagtc tggacctgag ctggtgaggc
ctggggcttc agtgaagctg 60 tcctgcaagg cttctggcta cacgttcacc
agctactgga tgaactgggt tcagcagagg 120 cctgagcaag gccttgagtg
gattggaagg attgatcctt acgatagtga aactcactac 180 aatcaaaagt
tcaaggacaa ggccatattg actgtagaca aatccgccag cacagcctac 240
atgcaactca gcagcctgac atctgaggac tctgcggtct attactgtgc aaaaatgggt
300 gattactcct ttgactactg gggccaaggg accacggtca ccgtctcctc a 351 13
117 PRT Artificial Sequence Description of Artificial Sequence
Synthetic Anti-NKG2D hybridoma 11B2D10 variable heavy chain 13 Gln
Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Arg Pro Gly Ala 1 5 10
15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Trp Met Asn Trp Val Gln Gln Arg Pro Glu Gln Gly Leu Glu
Trp Ile 35 40 45 Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr
Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Ile Leu Thr Val Asp Lys
Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser
Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Met Gly Asp Tyr
Ser Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser
Ser 115 14 10 PRT Artificial Sequence Description of Artificial
Sequence Synthetic Anti-NKG2D hybridoma 11B2D10 variable heavy
chain CDR1 14 Gly Tyr Thr Phe Thr Ser Tyr Trp Met Asn 1 5 10 15 17
PRT Artificial Sequence Description of Artificial Sequence
Synthetic Anti-NKG2D hybridoma 11B2D10 variable heavy chain CDR2 15
Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe Lys 1 5
10 15 Asp 16 8 PRT Artificial Sequence Description of Artificial
Sequence Synthetic Anti-NKG2D hybridoma 11B2D10 variable heavy
chain CDR3 16 Met Gly Asp Tyr Ser Phe Asp Tyr 1 5 17 318 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
Anti-NKG2D hybridoma 6H7E7 variable light chain 17 gacattcagc
tgacccagtc tccagcaatc ctgtctgcat ctccagggga gaaggtcaca 60
atgacttgca gggccagctc aagtgtaagt tacatgcact ggtaccagca gaagccagga
120 tcctccccca aaccctggat ttatgccaca tccaacctgg cttctggagt
ccctgctcgc 180 ttcagtggca gtgggtctgg gacctcttac tctctcacaa
tcagcagagt ggaggctgaa 240 gatgctgcca cttattactg ccagcagtgg
aatagtaacc cgctcacgtt cggtgctggg 300 accaagctgg agatcaaa 318 18 106
PRT Artificial Sequence Description of Artificial Sequence
Synthetic Anti-NKG2D hybridoma 6H7E7 variable light chain 18 Asp
Ile Gln Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly 1 5 10
15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met
20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp
Ile Tyr 35 40 45 Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg
Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile
Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln
Gln Trp Asn Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys
Leu Glu Ile Lys 100 105 19 10 PRT Artificial Sequence Description
of Artificial Sequence Synthetic Anti-NKG2D hybridoma 6H7E7
variable light chain CDR1 19 Arg Ala Ser Ser Ser Val Ser Tyr Met
His 1 5 10 20 7 PRT Artificial Sequence Description of Artificial
Sequence Synthetic Anti-NKG2D hybridoma 6H7E7 variable light chain
CDR2 20 Ala Thr Ser Asn Leu Ala Ser 1 5 21 9 PRT Artificial
Sequence Description of Artificial Sequence Synthetic Anti-NKG2D
hybridoma 6H7E7 variable light chain CDR3 21 Gln Gln Trp Asn Ser
Asn Pro Leu Thr 1 5 22 357 DNA Artificial Sequence Description of
Artificial Sequence Synthetic Anti-NKG2D hybridoma 6H7E7 variable
heavy chain 22 caggtgcagc tgcaggagtc aggacctggc ctggtggcgc
cctcacagag cctgtccatc 60 acttgcactg tctctgggtt ttcattaacc
agctatggtg tacactggat tcgccagcct 120 ccaggaaagg gtctggagtg
gctgggagta atatgggctg gtggaagcac aaattataat 180 tcggctctca
tgtccagact gagcatcagc aaagacaact ccaagagcca agttttctta 240
aaaatgaata gtctgcaaat tgatgacaca gccatgtact actgtgccag aggggggtac
300 gagggggcgg cctggtttgg ttactggggc caagggacca cggtcaccgt ctcctca
357 23 119 PRT Artificial Sequence Description of Artificial
Sequence Synthetic Anti-NKG2D hybridoma 6H7E7 variable heavy chain
23 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr
Ser Tyr 20 25 30 Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly
Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ala Gly Gly Ser Thr Asn
Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp
Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln
Ile Asp Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Arg Gly Gly Tyr
Glu Gly Ala Ala Trp Phe Gly Tyr Trp Gly Gln Gly 100 105 110 Thr Thr
Val Thr Val Ser Ser 115 24 10 PRT Artificial Sequence Description
of Artificial Sequence Synthetic Anti-NKG2D hybridoma 6H7E7
variable heavy chain CDR1 24 Gly Phe Ser Leu Thr Ser Tyr Gly Val
His 1 5 10 25 16 PRT Artificial Sequence Description of Artificial
Sequence Synthetic Anti-NKG2D hybridoma 6H7E7 variable heavy chain
CDR2 25 Val Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met
Ser 1 5 10 15 26 11 PRT Artificial Sequence Description of
Artificial Sequence Synthetic Anti-NKG2D hybridoma 6H7E7 variable
heavy chain CDR3 26 Gly Gly Tyr Glu Gly Ala Ala Trp Phe Gly Tyr 1 5
10 27 321 DNA Artificial Sequence Description of Artificial
Sequence Synthetic Anti-NKG2D hybridoma 8G7C10 variable light chain
27 gacattcagc tgacccagtc tccagccatc ctgtctgtga gtccaggaga
aagagtcagt 60 ttctcctgca gggccagtca gaccattggc acaagcattc
actggtatca gcaaagaaca 120 aatggttctc caaggcttct cataaagtat
gcttctgagt ctatctctgg gatcccttcc 180 aggtttagtg gcagtggatc
agggacagat tttactctta gcatcaacgg tgtggagtct 240 gaagatattg
cagattatta ctgtcaacaa agtaatacct ggccactcac gttcggtgct 300
gggaccaagc tggagatcaa a 321 28 107 PRT Artificial Sequence
Description of Artificial Sequence Synthetic Anti-NKG2D hybridoma
8G7C10 variable light chain 28 Asp Ile Gln Leu Thr Gln Ser Pro Ala
Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys
Arg Ala Ser Gln Thr Ile Gly Thr Ser 20 25 30 Ile His Trp Tyr Gln
Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala
Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Gly Val Glu Ser 65 70
75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser Asn Thr Trp Pro
Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys 100 105 29
11 PRT Artificial Sequence Description of Artificial Sequence
Synthetic Anti-NKG2D hybridoma 8G7C10 variable light chain CDR1 29
Arg Ala Ser Gln Thr Ile Gly Thr Ser Ile His 1 5 10 30 7 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
Anti-NKG2D hybridoma 8G7C10 variable light chain CDR2 30 Tyr Ala
Ser Glu Ser Ile Ser 1 5 31 9 PRT Artificial Sequence Description of
Artificial Sequence Synthetic Anti-NKG2D hybridoma 8G7C10 variable
light chain CDR3 31 Gln Gln Ser Asn Thr Trp Pro Leu Thr 1 5 32 360
DNA Artificial Sequence Description of Artificial Sequence
Synthetic Anti-NKG2D hybridoma 8G7C10 variable heavy chain 32
caggtgcagc tgcagcagtc aggacctggc ctagtgcagc cctcacagag cctgtccatc
60 acctgcacag tctctggttt ctcattaact atctatggtg tacactgggt
tcgccagtct 120 ccaggaaagg gtctggagtg gctgggagtg atatggagtg
gcggaagcac agactataat 180 gcagctttca tatccagact gagcatcagc
aaggacaatt ccaagcgcca agttttcttt 240 aaaatgagca gtctgcaagc
taatgacaca gccatatatt actgttccag aaagtcccat 300 gatggttact
acggagtaat ggactactgg ggccaaggga ccacggtcac cgtctcctca 360 33 120
PRT Artificial Sequence Description of Artificial Sequence
Synthetic Anti-NKG2D hybridoma 8G7C10 variable heavy chain 33 Gln
Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10
15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ile Tyr
20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu
Trp Leu 35 40 45 Gly Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn
Ala Ala Phe Ile 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser
Lys Arg Gln Val Phe Phe 65 70 75 80 Lys Met Ser Ser Leu Gln Ala Asn
Asp Thr Ala Ile Tyr Tyr Cys Ser 85 90 95 Arg Lys Ser His Asp Gly
Tyr Tyr Gly Val Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val
Thr Val Ser Ser 115 120 34 10 PRT Artificial Sequence Description
of Artificial Sequence Synthetic Anti-NKG2D hybridoma 8G7C10
variable heavy chain CDR1 34 Gly Phe Ser Leu Thr Ile Tyr Gly Val
His 1 5 10 35 16 PRT Artificial Sequence Description of Artificial
Sequence Synthetic Anti-NKG2D hybridoma 8G7C10 variable heavy chain
CDR2 35 Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile
Ser 1 5 10 15 36 12 PRT Artificial Sequence Description of
Artificial Sequence Synthetic Anti-NKG2D hybridoma 8G7C10 variable
heavy chain CDR3 36 Lys Ser His Asp Gly Tyr Tyr Gly Val Met Asp Tyr
1 5 10 37 321 DNA Artificial Sequence Description of Artificial
Sequence Synthetic Anti-NKG2D hybridoma 6E5A7 variable light chain
37 gacattcagc tgacccagtc tccagccatc ctgtctgtga gtccaggaga
aagagtcagt 60 ttctcctgca gggccagtca gagcattggc acaagcattc
actggtatca gcaaagaaca 120 aatggttctc caaggcttct cataaagtat
gcttctgagt ctatctctgg gatcccttcc 180 aggtttagtg gcagtggatc
agggacagat tttactctta gcatcaacgg tgtggagtct 240 gaagatattg
cagattatta ctgtcaacaa agtaatacct ggccactcac gttcggtgct 300
gggaccaagc tggagatcaa a 321 38 107 PRT Artificial Sequence
Description of Artificial Sequence Synthetic Anti-NKG2D hybridoma
6E5A7 variable light chain 38 Asp Ile Gln Leu Thr Gln Ser Pro Ala
Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys
Arg Ala Ser Gln Ser Ile Gly Thr Ser 20 25
30 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile
35 40 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn
Gly Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln
Ser Asn Thr Trp Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu
Glu Ile Lys 100 105 39 11 PRT Artificial Sequence Description of
Artificial Sequence Synthetic Anti-NKG2D hybridoma 6E5A7 variable
light chain CDR1 39 Arg Ala Ser Gln Ser Ile Gly Thr Ser Ile His 1 5
10 40 7 PRT Artificial Sequence Description of Artificial Sequence
Synthetic Anti-NKG2D hybridoma 6E5A7 variable light chain CDR2 40
Tyr Ala Ser Glu Ser Ile Ser 1 5 41 9 PRT Artificial Sequence
Description of Artificial Sequence Synthetic Anti-NKG2D hybridoma
6E5A7 variable light chain CDR3 41 Gln Gln Ser Asn Thr Trp Pro Leu
Thr 1 5 42 360 DNA Artificial Sequence Description of Artificial
Sequence Synthetic Anti-NKG2D hybridoma 6E5A7 variable heavy chain
42 caggtgcagc tgcagcagtc aggacctggc ctagtgcagc cctcacagag
cctgtccatc 60 acctgcacag tctctggttt ctcattaact atctatggtg
tacactgggt tcgccagtct 120 ccaggaaagg gtctggagtg gctgggagtg
atatggagtg gcggaagcac agactataat 180 gcagctttca tatccagact
gagcatcagc aaggacaatt ccaagcgcca agttttcttt 240 aaaatgagca
gtctgcaagc taatgacaca gccatatatt actgttccag aaagtcccat 300
gatggttact acggagtaat ggactactgg ggccaaggga ccacggtcac cgtctcctca
360 43 120 PRT Artificial Sequence Description of Artificial
Sequence Synthetic Anti-NKG2D hybridoma 6E5A7 variable heavy chain
43 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln
1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr
Ile Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly
Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ser Gly Gly Ser Thr Asp
Tyr Asn Ala Ala Phe Ile 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp
Asn Ser Lys Arg Gln Val Phe Phe 65 70 75 80 Lys Met Ser Ser Leu Gln
Ala Asn Asp Thr Ala Ile Tyr Tyr Cys Ser 85 90 95 Arg Lys Ser His
Asp Gly Tyr Tyr Gly Val Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr
Thr Val Thr Val Ser Ser 115 120 44 10 PRT Artificial Sequence
Description of Artificial Sequence Synthetic Anti-NKG2D hybridoma
6E5A7 variable heavy chain CDR1 44 Gly Phe Ser Leu Thr Ile Tyr Gly
Val His 1 5 10 45 16 PRT Artificial Sequence Description of
Artificial Sequence Synthetic Anti-NKG2D hybridoma 6E5A7 variable
heavy chain CDR2 45 Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala
Ala Phe Ile Ser 1 5 10 15 46 12 PRT Artificial Sequence Description
of Artificial Sequence Synthetic Anti-NKG2D hybridoma 6E5A7
variable heavy chain CDR3 46 Lys Ser His Asp Gly Tyr Tyr Gly Val
Met Asp Tyr 1 5 10 47 507 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 11B2D10x4- 7 bispecific single chain
Fv 47 Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val
Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile
His Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser
Pro Gln Val Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Asp Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Tyr
Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser
Tyr Tyr Cys Gln His Phe Trp Ser Thr Thr Trp 85 90 95 Thr Phe Gly
Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln 115 120
125 Ser Gly Pro Glu Leu Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys
130 135 140 Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Asn Trp
Val Gln 145 150 155 160 Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly
Arg Ile Asp Pro Tyr 165 170 175 Asp Ser Glu Thr His Tyr Asn Gln Lys
Phe Lys Asp Lys Ala Ile Leu 180 185 190 Thr Val Asp Lys Ser Ala Ser
Thr Ala Tyr Met Gln Leu Ser Ser Leu 195 200 205 Thr Ser Glu Asp Ser
Ala Val Tyr Tyr Cys Ala Lys Met Gly Asp Tyr 210 215 220 Ser Phe Asp
Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly 225 230 235 240
Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu 245
250 255 Ala Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly
Tyr 260 265 270 Thr Phe Thr Asn Tyr Gly Leu Ser Trp Val Lys Gln Arg
Pro Gly Gln 275 280 285 Val Leu Glu Trp Ile Gly Glu Val Tyr Pro Arg
Ile Gly Asn Ala Tyr 290 295 300 Tyr Asn Glu Lys Phe Lys Gly Lys Ala
Thr Leu Thr Ala Asp Lys Ser 305 310 315 320 Ser Ser Thr Ala Ser Met
Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser 325 330 335 Ala Val Tyr Phe
Cys Ala Arg Arg Gly Ser Tyr Asp Thr Asn Tyr Asp 340 345 350 Trp Tyr
Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 355 360 365
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 370
375 380 Leu Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly
Asp 385 390 395 400 Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu
Val His Ser Asn 405 410 415 Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln
Lys Pro Gly Gln Ser Pro 420 425 430 Lys Leu Leu Ile Tyr Lys Val Ser
Asn Arg Phe Ser Gly Val Pro Asp 435 440 445 Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser 450 455 460 Arg Val Glu Ala
Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser Thr 465 470 475 480 His
Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 485 490
495 Thr Thr Ser His His His His His His Thr Ser 500 505 48 510 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
8G7C10x4-7 bispecific single chain Fv 48 Asp Ile Gln Leu Thr Gln
Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser
Phe Ser Cys Arg Ala Ser Gln Thr Ile Gly Thr Ser 20 25 30 Ile His
Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45
Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Gly Val Glu
Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser Asn Thr
Trp Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys
Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gln Val Gln Leu Gln Gln 115 120 125 Ser Gly Pro Gly Leu Val Gln
Pro Ser Gln Ser Leu Ser Ile Thr Cys 130 135 140 Thr Val Ser Gly Phe
Ser Leu Thr Ile Tyr Gly Val His Trp Val Arg 145 150 155 160 Gln Ser
Pro Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly 165 170 175
Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile Ser Arg Leu Ser Ile Ser 180
185 190 Lys Asp Asn Ser Lys Arg Gln Val Phe Phe Lys Met Ser Ser Leu
Gln 195 200 205 Ala Asn Asp Thr Ala Ile Tyr Tyr Cys Ser Arg Lys Ser
His Asp Gly 210 215 220 Tyr Tyr Gly Val Met Asp Tyr Trp Gly Gln Gly
Thr Thr Val Thr Val 225 230 235 240 Ser Ser Gly Gly Gly Gly Ser Glu
Val Gln Leu Leu Glu Gln Ser Gly 245 250 255 Ala Glu Leu Ala Arg Pro
Gly Ala Ser Val Lys Leu Ser Cys Lys Ala 260 265 270 Ser Gly Tyr Thr
Phe Thr Asn Tyr Gly Leu Ser Trp Val Lys Gln Arg 275 280 285 Pro Gly
Gln Val Leu Glu Trp Ile Gly Glu Val Tyr Pro Arg Ile Gly 290 295 300
Asn Ala Tyr Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala 305
310 315 320 Asp Lys Ser Ser Ser Thr Ala Ser Met Glu Leu Arg Ser Leu
Thr Ser 325 330 335 Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Arg Gly
Ser Tyr Asp Thr 340 345 350 Asn Tyr Asp Trp Tyr Phe Asp Val Trp Gly
Gln Gly Thr Thr Val Thr 355 360 365 Val Ser Ser Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly 370 375 380 Gly Ser Glu Leu Val Met
Thr Gln Thr Pro Leu Ser Leu Pro Val Ser 385 390 395 400 Leu Gly Asp
Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val 405 410 415 His
Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly 420 425
430 Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
435 440 445 Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu 450 455 460 Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val
Tyr Phe Cys Ser 465 470 475 480 Gln Ser Thr His Val Pro Tyr Thr Phe
Gly Gly Gly Thr Lys Leu Glu 485 490 495 Ile Lys Arg Thr Thr Ser His
His His His His His Thr Ser 500 505 510 49 510 PRT Artificial
Sequence Description of Artificial Sequence Synthetic 6E5A7x4-7
bispecific single chain Fv 49 Asp Ile Gln Leu Thr Gln Ser Pro Ala
Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys
Arg Ala Ser Gln Ser Ile Gly Thr Ser 20 25 30 Ile His Trp Tyr Gln
Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala
Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Gly Val Glu Ser 65 70
75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser Asn Thr Trp Pro
Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Gly Gly
Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
Val Gln Leu Gln Gln 115 120 125 Ser Gly Pro Gly Leu Val Gln Pro Ser
Gln Ser Leu Ser Ile Thr Cys 130 135 140 Thr Val Ser Gly Phe Ser Leu
Thr Ile Tyr Gly Val His Trp Val Arg 145 150 155 160 Gln Ser Pro Gly
Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly 165 170 175 Gly Ser
Thr Asp Tyr Asn Ala Ala Phe Ile Ser Arg Leu Ser Ile Ser 180 185 190
Lys Asp Asn Ser Lys Arg Gln Val Phe Phe Lys Met Ser Ser Leu Gln 195
200 205 Ala Asn Asp Thr Ala Ile Tyr Tyr Cys Ser Arg Lys Ser His Asp
Gly 210 215 220 Tyr Tyr Gly Val Met Asp Tyr Trp Gly Gln Gly Thr Thr
Val Thr Val 225 230 235 240 Ser Ser Gly Gly Gly Gly Ser Glu Val Gln
Leu Leu Glu Gln Ser Gly 245 250 255 Ala Glu Leu Ala Arg Pro Gly Ala
Ser Val Lys Leu Ser Cys Lys Ala 260 265 270 Ser Gly Tyr Thr Phe Thr
Asn Tyr Gly Leu Ser Trp Val Lys Gln Arg 275 280 285 Pro Gly Gln Val
Leu Glu Trp Ile Gly Glu Val Tyr Pro Arg Ile Gly 290 295 300 Asn Ala
Tyr Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala 305 310 315
320 Asp Lys Ser Ser Ser Thr Ala Ser Met Glu Leu Arg Ser Leu Thr Ser
325 330 335 Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Arg Gly Ser Tyr
Asp Thr 340 345 350 Asn Tyr Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly
Thr Thr Val Thr 355 360 365 Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly 370 375 380 Gly Ser Glu Leu Val Met Thr Gln
Thr Pro Leu Ser Leu Pro Val Ser 385 390 395 400 Leu Gly Asp Gln Ala
Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val 405 410 415 His Ser Asn
Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly 420 425 430 Gln
Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly 435 440
445 Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
450 455 460 Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe
Cys Ser 465 470 475 480 Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly
Gly Thr Lys Leu Glu 485 490 495 Ile Lys Arg Thr Thr Ser His His His
His His His Thr Ser 500 505 510 50 206 DNA Homo sapiens 50
actagttccg gaaccccgct gggtgacacc acccacacct ctggaaaacc actggatgga
60 gaatatttca cccttcagat ccgtgggcgt gagcgcttcg agatgttccg
agagctgaat 120 gaggccttgg aactcaagga tgcccaggct gggaaggagc
caggggggag cgactacaag 180 gatgacgatg acaagtaagc ggccgc 206 51 65
PRT Homo sapiens 51 Thr Ser Ser Gly Thr Pro Leu Gly Asp Thr Thr His
Thr Ser Gly Lys 1 5 10 15 Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln
Ile Arg Gly Arg Glu Arg 20 25 30 Phe Glu Met Phe Arg Glu Leu Asn
Glu Ala Leu Glu Leu Lys Asp Ala 35 40 45 Gln Ala Gly Lys Glu Pro
Gly Gly Ser Asp Tyr Lys Asp Asp Asp Asp 50 55 60 Lys 65 52 777 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
P4-2 single chain Fv 52 gaggtgcagc tgctcgagga gtctggagga ggcttggtac
agcctggggg ttctctgaga 60 ctctcctgtg caacttctgg gttcaccttc
actgattact acatgagctg ggtccgccag 120 cctccaggaa aggcacttga
gtggttgggt tttattagaa acaaagctaa tggttacaca 180 acagagtaca
gtgcatctgt gaagggtcgg ttcaccatct ccagagataa ttcccaaagc 240
atcctctatc ttcaaatgaa caccctgaga gctgaggaca gtcccactta ttactgtgca
300 agagataaga cagacttcga tgtctggggc caagggacca cggtcaccgt
ctcctcaggt 360 ggtggtggtt ctggcggcgg cggctccggt ggtggtggtt
ctgagctcgt gatgacacag 420 tctccatcct ccctgactgt gacagcagga
gagaaggtca ctatgagctg caagtccagt 480 cagagtctgt taaacagtgg
aaatcaaaag aactacttga cctggtacca gcagaaacca 540 gggcagcctc
ctaaactgtt gatctactgg gcatccacta gggaatctgg ggtccctgat 600
cgcttcacag gcagtggatc tggaacagat ttcactctca ccatcagcag tgtgcaggct
660 gaagacctgg cagtttatta ctgtcagaat gattatagtt atccgctcac
gttcggtgct 720 gggaccaagc ttgagatcaa acgtacgact agttccgggc
atcatcacca tcatcat 777 53 259 PRT Artificial Sequence Description
of Artificial Sequence Synthetic P4-2 single chain Fv 53 Glu Val
Gln Leu Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp 20
25 30 Tyr Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu
Trp 35 40 45 Leu Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr
Glu Tyr Ser 50 55 60 Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Gln Ser 65 70 75 80 Ile Leu Tyr Leu Gln Met Asn Thr Leu
Arg Ala Glu Asp Ser Pro Thr 85 90 95 Tyr Tyr Cys Ala Arg Asp Lys
Thr Asp Phe Asp Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly
Gly Gly Ser Glu
Leu Val Met Thr Gln Ser Pro Ser Ser 130 135 140 Leu Thr Val Thr Ala
Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser 145 150 155 160 Gln Ser
Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr 165 170 175
Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser 180
185 190 Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser
Gly 195 200 205 Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu
Asp Leu Ala 210 215 220 Val Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro
Leu Thr Phe Gly Ala 225 230 235 240 Gly Thr Lys Leu Glu Ile Lys Arg
Thr Thr Ser Ser Gly His His His 245 250 255 His His His 54 756 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
P4-3 single chain Fv 54 gaggtgcagc tgctcgagtc tggaggtggc ctggtgcagc
ctggaggatc cctgaaactc 60 tcctgtgcag cctctggatt cgattttagt
agatactgga tgagttgggt ccggcaggct 120 ccagggaaag ggctagaatg
gattggagaa attaatccag atagcagtac gataaactat 180 acgccatctc
taaaggataa attcatcatc tccagagaca acgccaaaaa tacgctgtac 240
ctgcaaatga gcaaagtgag atctgaggac acagcccttt attactgtgc aagaggggcg
300 gtagtagctc cctttgacta ctggggccaa gggaccacgg tcaccgtctc
ctcaggtggt 360 ggtggttctg gcggcggcgg ctccggtggt ggtggttctg
agctcgtcat gacccagtct 420 ccatcctcct tatctgcctc tctgggagaa
agagtcagtc tcacttgtcg ggcaagtcag 480 gacattggta gtagcttaaa
ctggcttcag caggaaccag atggaactat taaacgcctg 540 atctacgcca
catccagttt agattctggt gtccccaaaa ggttcagtgg cagtaggtct 600
gggtcagatt attctctcac catcagcagc cttgagtctg aagattttgt agactattac
660 tgtctacaat atgctagttc tccgtacacg ttcggagggg ggaccaagct
tgagatcaaa 720 cgtacgacta gttccgggca tcatcaccat catcat 756 55 252
PRT Artificial Sequence Description of Artificial Sequence
Synthetic P4-3 single chain Fv 55 Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys
Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu
Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu 50 55 60
Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Ser Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr
Tyr Cys 85 90 95 Ala Arg Gly Ala Val Val Ala Pro Phe Asp Tyr Trp
Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Glu Leu Val
Met Thr Gln Ser Pro Ser Ser Leu 130 135 140 Ser Ala Ser Leu Gly Glu
Arg Val Ser Leu Thr Cys Arg Ala Ser Gln 145 150 155 160 Asp Ile Gly
Ser Ser Leu Asn Trp Leu Gln Gln Glu Pro Asp Gly Thr 165 170 175 Ile
Lys Arg Leu Ile Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro 180 185
190 Lys Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile
195 200 205 Ser Ser Leu Glu Ser Glu Asp Phe Val Asp Tyr Tyr Cys Leu
Gln Tyr 210 215 220 Ala Ser Ser Pro Tyr Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 225 230 235 240 Arg Thr Thr Ser Ser Gly His His His
His His His 245 250 56 768 DNA Artificial Sequence Description of
Artificial Sequence Synthetic P4-14 single chain Fv 56 gaggtgcagc
tgctcgagtc tggaggtggc ctggtgcagc ctggaggatc cctgaaactc 60
tcctgtgcag cctcaggatt cgattttagt agatactgga tgagttgggt ccggcaggct
120 ccagggaaag ggctagaatg gattggagaa attaatccag atagcagtac
gataaactat 180 acgccatctc taaaggataa attcatcatc tccagagaca
acgccaaaaa tacgctgtac 240 ctgcaaatga gcaaagtgag atctgaggac
acagcccttt attactgtgc aagacgcagc 300 tacggtagta gctacgactg
gtacttcgat gtctggggcc aagggaccac ggtcaccgtc 360 tcctcaggtg
gtggtggttc tggcggcggc ggctccggtg gtggtggttc tgagctccag 420
atgacccagt ctccagcctc cctatctgca tctgtgggag aaactgtcac catcacatgt
480 cgagcaagtg agaatattta cagttattta gcatggtatc agcagaaaca
gggaaaatct 540 cctcagctcc tggtctataa tgcaaaaacc ttagcagaag
gtgtgccatc aaggttcagt 600 agcagtggat caggcacaca gttttctctg
aagatcaaca gcctgcagcc tgaagatttt 660 gggagttatt actgtcaaca
tcattatggt actccgctca cgttcggtgc tgggaccaag 720 cttgagatca
aacgtacgac tagttccggg catcatcacc atcatcat 768 57 256 PRT Artificial
Sequence Description of Artificial Sequence Synthetic P4-14 single
chain Fv 57 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp
Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Asp Ser Ser
Thr Ile Asn Tyr Thr Pro Ser Leu 50 55 60 Lys Asp Lys Phe Ile Ile
Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser
Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Arg
Arg Ser Tyr Gly Ser Ser Tyr Asp Trp Tyr Phe Asp Val Trp 100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 115
120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Gln Met Thr Gln
Ser 130 135 140 Pro Ala Ser Leu Ser Ala Ser Val Gly Glu Thr Val Thr
Ile Thr Cys 145 150 155 160 Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu
Ala Trp Tyr Gln Gln Lys 165 170 175 Gln Gly Lys Ser Pro Gln Leu Leu
Val Tyr Asn Ala Lys Thr Leu Ala 180 185 190 Glu Gly Val Pro Ser Arg
Phe Ser Ser Ser Gly Ser Gly Thr Gln Phe 195 200 205 Ser Leu Lys Ile
Asn Ser Leu Gln Pro Glu Asp Phe Gly Ser Tyr Tyr 210 215 220 Cys Gln
His His Tyr Gly Thr Pro Leu Thr Phe Gly Ala Gly Thr Lys 225 230 235
240 Leu Glu Ile Lys Arg Thr Thr Ser Ser Gly His His His His His His
245 250 255 58 774 DNA Artificial Sequence Description of
Artificial Sequence Synthetic P4-15 single chain Fv 58 gaggtgcagc
tgctcgagca gtctggagct gagctgatga agcctggggc ctcagtgaag 60
atatcctgca aggctactgg ctacacattc agtagctact ggatagagtg ggtaaagcag
120 aggcctggac atggccttga gtggattgga gagattttac ctggaagtgg
tagtactaac 180 tacaatgaga agttcaaggg caaggccaca ttcactgcag
atacatcctc caacacagcc 240 tacatgcaac tcagcagcct gacatctgag
gactctgccg tctattactg tgcaagagga 300 ttacgacgtt ggtttgctta
ctggggccaa gggaccacgg tcaccgtctc ctcaggtggt 360 ggtggttctg
gcggcggcgg ctccggtggt ggtggttctg agctcgtgat gacacagtct 420
ccatcctccc tgactgtgac agcaggagag aaggtcacta tgagctgcaa gtccagtcag
480 agtctgttaa acagtggaaa tcaaaagaac tacttgacct ggtaccagca
gaaaccaggg 540 cagcctccta aactgttgat ctactgggca tccactaggg
aatctggggt ccctgatcgc 600 ttcacaggca gtggatctgg aacagatttc
actctcacca tcagcagtgt gcaggctgaa 660 gacctggcag tttattactg
tcagaatgat tatagttatc cgctcacgtt cggtgctggg 720 accaagcttg
agatcaaacg tacgactagt tccgggcatc atcaccatca tcat 774 59 258 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
P4-15 single chain Fv 59 Glu Val Gln Leu Leu Glu Gln Ser Gly Ala
Glu Leu Met Lys Pro Gly 1 5 10 15 Ala Ser Val Lys Ile Ser Cys Lys
Ala Thr Gly Tyr Thr Phe Ser Ser 20 25 30 Tyr Trp Ile Glu Trp Val
Lys Gln Arg Pro Gly His Gly Leu Glu Trp 35 40 45 Ile Gly Glu Ile
Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn Glu Lys 50 55 60 Phe Lys
Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala 65 70 75 80
Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr 85
90 95 Cys Ala Arg Gly Leu Arg Arg Trp Phe Ala Tyr Trp Gly Gln Gly
Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Glu Leu Val Met Thr Gln
Ser Pro Ser Ser Leu 130 135 140 Thr Val Thr Ala Gly Glu Lys Val Thr
Met Ser Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Leu Asn Ser Gly
Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly
Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu
Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr 195 200 205
Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val 210
215 220 Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala
Gly 225 230 235 240 Thr Lys Leu Glu Ile Lys Arg Thr Thr Ser Ser Gly
His His His His 245 250 255 His His 60 768 DNA Artificial Sequence
Description of Artificial Sequence Synthetic P5-2 single chain Fv
60 gaggtgcagc tgctcgagga gtctggagga ggcttggtgc aacctggagg
atccatgaaa 60 ctctcctgtg ttgcctctgg attcactttc agtaactact
ggatgaactg ggtccgccag 120 tctccagaga aggggcttga gtgggttgct
gaaattagat tgaaatctaa taattatgca 180 acacattatg cggagtctgt
gaaagggagg ttcaccatct caagagatga ttccaaaagt 240 agtgtctacc
tgcaaatgaa caacttaaga gctgaagaca ctggcattta ttactgtacc 300
aggctcccct acggctttgc tatggactac tggggccaag ggaccacggt caccgtctcc
360 tcaggtggtg gtggttctgg cggcggcggc tccggtggtg gtggttctga
gctcgtgctc 420 acccagtctc caaccaccat ggctgcatct cccggggaga
agatcactat cacctgcagt 480 gccagctcaa gtataagttc caattacttg
cattggtatc agcagaagcc aggattctcc 540 cctaaactct tgatttatag
gacatccaat ctggcttctg gagtcccagc tcgcttcagt 600 ggcagtgggt
ctgggacctc ttactctctc acaattggca ccatggaggc tgaagatgtt 660
gccacttact actgccagca gggtagtagt ataccgctca cgttcggtgc tgggaccaag
720 cttgagatca aacgtacgac tagttccggg catcatcacc atcatcat 768 61 256
PRT Artificial Sequence Description of Artificial Sequence
Synthetic P5-2 single chain Fv 61 Glu Val Gln Leu Leu Glu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Met Lys Leu Ser
Cys Val Ala Ser Gly Phe Thr Phe Ser Asn 20 25 30 Tyr Trp Met Asn
Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp 35 40 45 Val Ala
Glu Ile Arg Leu Lys Ser Asn Asn Tyr Ala Thr His Tyr Ala 50 55 60
Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser 65
70 75 80 Ser Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr
Gly Ile 85 90 95 Tyr Tyr Cys Thr Arg Leu Pro Tyr Gly Phe Ala Met
Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Gly
Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser
Glu Leu Val Leu Thr Gln Ser Pro 130 135 140 Thr Thr Met Ala Ala Ser
Pro Gly Glu Lys Ile Thr Ile Thr Cys Ser 145 150 155 160 Ala Ser Ser
Ser Ile Ser Ser Asn Tyr Leu His Trp Tyr Gln Gln Lys 165 170 175 Pro
Gly Phe Ser Pro Lys Leu Leu Ile Tyr Arg Thr Ser Asn Leu Ala 180 185
190 Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr
195 200 205 Ser Leu Thr Ile Gly Thr Met Glu Ala Glu Asp Val Ala Thr
Tyr Tyr 210 215 220 Cys Gln Gln Gly Ser Ser Ile Pro Leu Thr Phe Gly
Ala Gly Thr Lys 225 230 235 240 Leu Glu Ile Lys Arg Thr Thr Ser Ser
Gly His His His His His His 245 250 255 62 759 DNA Artificial
Sequence Description of Artificial Sequence Synthetic P5-3 single
chain Fv 62 gaggtgcagc tgctcgagga gtcaggacct ggcctggtgg cgccctcaca
gagcctgtcc 60 atcacttgca ctgtctctgg gttttcatta accagctatg
gtgtacactg ggttcgccag 120 cctccaggaa agggtctgga gtggctggga
gtaatatggg ctggtggaag cacaaattat 180 aattcggctc tcatgtccag
actgagcatc agcaaagaca actccaagag ccaagttttc 240 ttaaaaatga
acagtctgca aactgatgac acagccatgt actactgtgc cagagatcgg 300
tactacgtgg gtgctatgga ctactggggc caagggacca cggtcaccgt ctcctcaggt
360 ggtggtggtt ctggcggcgg cggctccggt ggtggtggtt ctgagctcca
gatgacccag 420 tctccagcat ccctgtccat ggctatagga gaaaaagtca
ccatcagatg cataaccagc 480 actgatattg atgatgatat gaactggtac
cagcagaagc caggggaacc tcctaagctc 540 cttatttcag aaggcaatac
tcttcgtcct ggagtcccat cccgattctc cagcagtggc 600 tatggtacag
attttgtttt tacaattgaa aacatgctct cagaagatgt tgcagattac 660
tactgtttgc aaagtgataa cttgccgtac acgttcggag gggggaccaa gcttgagatc
720 aaacgtacga ctagttccgg gcatcatcac catcatcat 759 63 253 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
P5-3 single chain Fv 63 Glu Val Gln Leu Leu Glu Glu Ser Gly Pro Gly
Leu Val Ala Pro Ser 1 5 10 15 Gln Ser Leu Ser Ile Thr Cys Thr Val
Ser Gly Phe Ser Leu Thr Ser 20 25 30 Tyr Gly Val His Trp Val Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Leu Gly Val Ile Trp
Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu 50 55 60 Met Ser Arg
Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe 65 70 75 80 Leu
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys 85 90
95 Ala Arg Asp Arg Tyr Tyr Val Gly Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110 Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Glu Leu Gln Met Thr Gln
Ser Pro Ala Ser 130 135 140 Leu Ser Met Ala Ile Gly Glu Lys Val Thr
Ile Arg Cys Ile Thr Ser 145 150 155 160 Thr Asp Ile Asp Asp Asp Met
Asn Trp Tyr Gln Gln Lys Pro Gly Glu 165 170 175 Pro Pro Lys Leu Leu
Ile Ser Glu Gly Asn Thr Leu Arg Pro Gly Val 180 185 190 Pro Ser Arg
Phe Ser Ser Ser Gly Tyr Gly Thr Asp Phe Val Phe Thr 195 200 205 Ile
Glu Asn Met Leu Ser Glu Asp Val Ala Asp Tyr Tyr Cys Leu Gln 210 215
220 Ser Asp Asn Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
225 230 235 240 Lys Arg Thr Thr Ser Ser Gly His His His His His His
245 250 64 753 DNA Artificial Sequence Description of Artificial
Sequence Synthetic P5-9 single chain Fv 64 gaggtgcagc tgctcgagtc
tggaggtggc ctggtgcagc ctggaggatc cctgaaactc 60 tcctgtgcag
cctcaggatt cgattttagt agatactgga tgagttgggt ccggcaggct 120
ccagggaaag ggctagaatg gattggagaa attaatccag atagcagtac gataaactat
180 acgccatctc taaaggataa attcatcatc tccagagaca acgccaaaaa
tacgctgtac 240 ctgcaaatga gcaaagtgag atctgaggac acagcccttt
attactgtgc aagggaaact 300 gggacggagt ttgactactg gggccaaggg
accacggtca ccgtctcctc aggtggtggt 360 ggttctggcg gcggcggctc
cggtggtggt ggttctgagc tcgtgatgac ccagactcca 420 tcctccatgt
atgcatcgct gggagagaga gtcactatca cttgcaaggc gagtcaggac 480
attaaaagct atttaagctg gtaccagcag aaaccatgga aatctcctaa gaccctgatc
540 tattatgcaa caagcttggc agatggggtc ccatcaagat tcagtggcag
tggatctggg 600 caagattatt ctctaaccat cagcagcctg gagtctgacg
atacagcaac ttattactgt 660 ctacagcatg gtgagagccc gtacacgttc
ggagggggga ccaagcttga gatcaaacgt 720 acgactagtt ccgggcatca
tcaccatcat cat 753 65 251 PRT Artificial Sequence Description of
Artificial Sequence Synthetic P5-9 single chain Fv 65 Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr 20 25
30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45 Gly Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr Pro
Ser Leu 50 55 60 Lys Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Lys Val Arg Ser Glu Asp
Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Arg Glu Thr Gly Thr Glu Phe
Asp Tyr Trp Gly Gln Gly Thr Thr 100 105
110 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
115 120 125 Gly Gly Gly Ser Glu Leu Val Met Thr Gln Thr Pro Ser Ser
Met Tyr 130 135 140 Ala Ser Leu Gly Glu Arg Val Thr Ile Thr Cys Lys
Ala Ser Gln Asp 145 150 155 160 Ile Lys Ser Tyr Leu Ser Trp Tyr Gln
Gln Lys Pro Trp Lys Ser Pro 165 170 175 Lys Thr Leu Ile Tyr Tyr Ala
Thr Ser Leu Ala Asp Gly Val Pro Ser 180 185 190 Arg Phe Ser Gly Ser
Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser 195 200 205 Ser Leu Glu
Ser Asp Asp Thr Ala Thr Tyr Tyr Cys Leu Gln His Gly 210 215 220 Glu
Ser Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 225 230
235 240 Thr Thr Ser Ser Gly His His His His His His 245 250 66 771
DNA Artificial Sequence Description of Artificial Sequence
Synthetic P5-10 single chain Fv 66 gaggtgcagc tgctcgagca gtctggagct
gagcttgtga ggccaggggc cttagtcaag 60 ttgtcctgca aagcttctgg
cttcaacatt aaagactact atatgcactg ggtgaagcag 120 aggcctgaac
agggcctgga gtggattgga tggattgatc ctgagaatgg taatactata 180
tatgacccga agttccaggg caaggccagt ataacagcag acacatcctc caacacagcc
240 tacctgcagc tcagcagcct gacatctgag gacactgccg cctattactg
tgcttccttt 300 tattactacg gtagtagcta caggtacttc gatgtctggg
gccaagggac cacggtcacc 360 gtctcctcag gtggtggtgg ttctggcggc
ggcggctccg gtggtggtgg ttctgagctc 420 gtgatgaccc agactccatc
ctccttatct gcctctctgg gagaaagagt cagtctcact 480 tgtcgggcaa
gtcaggacat tggtagtagc ttaaactggc ttcagcagga accagatgga 540
actattaaac gcctgatcta cgccacatcc agtttagatt ctggtgtccc caaaaggttc
600 agtggcagta ggtctgggtc agattattct ctcaccatca gcagccttga
gtctgaagat 660 tttgtagact attactgtct acaatatgct agttctccgt
acacgttcgg aggggggacc 720 aagcttgaga tcaaacgtac gactagttcc
gggcatcatc accatcatca t 771 67 257 PRT Artificial Sequence
Description of Artificial Sequence Synthetic P5-10 single chain Fv
67 Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val Arg Pro Gly
1 5 10 15 Ala Leu Val Lys Leu Ser Cys Lys Ala Ser Gly Phe Asn Ile
Lys Asp 20 25 30 Tyr Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln
Gly Leu Glu Trp 35 40 45 Ile Gly Trp Ile Asp Pro Glu Asn Gly Asn
Thr Ile Tyr Asp Pro Lys 50 55 60 Phe Gln Gly Lys Ala Ser Ile Thr
Ala Asp Thr Ser Ser Asn Thr Ala 65 70 75 80 Tyr Leu Gln Leu Ser Ser
Leu Thr Ser Glu Asp Thr Ala Ala Tyr Tyr 85 90 95 Cys Ala Ser Phe
Tyr Tyr Tyr Gly Ser Ser Tyr Arg Tyr Phe Asp Val 100 105 110 Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Met Thr Gln 130
135 140 Thr Pro Ser Ser Leu Ser Ala Ser Leu Gly Glu Arg Val Ser Leu
Thr 145 150 155 160 Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser Leu Asn
Trp Leu Gln Gln 165 170 175 Glu Pro Asp Gly Thr Ile Lys Arg Leu Ile
Tyr Ala Thr Ser Ser Leu 180 185 190 Asp Ser Gly Val Pro Lys Arg Phe
Ser Gly Ser Arg Ser Gly Ser Asp 195 200 205 Tyr Ser Leu Thr Ile Ser
Ser Leu Glu Ser Glu Asp Phe Val Asp Tyr 210 215 220 Tyr Cys Leu Gln
Tyr Ala Ser Ser Pro Tyr Thr Phe Gly Gly Gly Thr 225 230 235 240 Lys
Leu Glu Ile Lys Arg Thr Thr Ser Ser Gly His His His His His 245 250
255 His 68 765 DNA Artificial Sequence Description of Artificial
Sequence Synthetic P5-11 single chain Fv 68 gaggtgcagc tgctcgagga
gtctggagga ggcttggtgc aacctggagg atccatgaaa 60 ctctcctgta
ttgcctctgg attcactttc agtaattcct ggatgaactg ggtccgccag 120
tctccagaga aggggcttga gtgggttggt gaaattagat tgaaatctaa taattatgca
180 acacattatg cggagtctgt gaaagggagg ttcaccatct caagagatga
ttccaaaagt 240 agtgtctacc tacaaatgaa caacttaaga gttgaagaca
ctggcattta ttactgtacg 300 aaggtggact actggggcca agggaccacg
gtcaccgtct cctcaggtgg tggtggttct 360 ggcggcggcg gctccggtgg
tggtggttct gagctcgtga tgacacagtc tccatcctcc 420 ctggctatgt
cagtaggaca gaaggtcact atgagctgca agtccagtca gagcctttta 480
aatagtagca atcaaaagaa ctacttgacc tggtaccagc agaaaccagg gcagcctcct
540 aaactgttga tctactgggc atccactagg gaatctgggg tccctgatcg
cttcacaggc 600 agtggatctg gaacagattt cactctcacc atcagcagtg
tgcaggctga agacctggca 660 gtttattact gtcagaatga ttatagttat
ccgctcacgt tcggtgctgg gaccaagctt 720 gagatcaaac gtacgactag
ttccgggcat catcaccatc atcat 765 69 255 PRT Artificial Sequence
Description of Artificial Sequence Synthetic P5-11 single chain Fv
69 Glu Val Gln Leu Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
1 5 10 15 Gly Ser Met Lys Leu Ser Cys Ile Ala Ser Gly Phe Thr Phe
Ser Asn 20 25 30 Ser Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys
Gly Leu Glu Trp 35 40 45 Val Gly Glu Ile Arg Leu Lys Ser Asn Asn
Tyr Ala Thr His Tyr Ala 50 55 60 Glu Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asp Ser Lys Ser 65 70 75 80 Ser Val Tyr Leu Gln Met
Asn Asn Leu Arg Val Glu Asp Thr Gly Ile 85 90 95 Tyr Tyr Cys Thr
Lys Val Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr 100 105 110 Val Ser
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125
Gly Ser Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ala Met Ser 130
135 140 Val Gly Gln Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu
Leu 145 150 155 160 Asn Ser Ser Asn Gln Lys Asn Tyr Leu Thr Trp Tyr
Gln Gln Lys Pro 165 170 175 Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp
Ala Ser Thr Arg Glu Ser 180 185 190 Gly Val Pro Asp Arg Phe Thr Gly
Ser Gly Ser Gly Thr Asp Phe Thr 195 200 205 Leu Thr Ile Ser Ser Val
Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys 210 215 220 Gln Asn Asp Tyr
Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu 225 230 235 240 Glu
Ile Lys Arg Thr Thr Ser Ser Gly His His His His His His 245 250 255
70 774 DNA Artificial Sequence Description of Artificial Sequence
Synthetic P5-23 single chain Fv 70 gaggtgcagc tgctcgagtc tggaggtggc
ctggtgcagc ctggaggatc cctgaaactc 60 tcctgtgcag cctcaggatt
cgattttagt agatactgga tgagttgggt ccggcaggct 120 ccagggaaag
ggctagaatg gattggagaa attaatccag atagcagtac gataaactat 180
acgccatctc taaaggatag attcatcatc tccagagaca acgccaaaaa tacgctgtac
240 ctgcaaatga gcaaagtgag gtctgaggac acagcccttt attactgtgc
aagattgggg 300 caatgggggt actttgacta ctggggccaa gggaccacgg
tcaccgtctc ctcaggtggt 360 ggtggttctg gcggcggcgg ctccggtggt
ggtggttctg agctcgtgat gacacagtct 420 ccatcctccc tgactgtgac
agcaggagag agggtcacta tgagctgcaa gtccagtcag 480 agtctgttaa
acagtggaaa tcaaaagaac tacttgacct ggtaccagca gaaaccaggg 540
cagcctccta aactgttgat ctactgggca tccactaggg aatctggggt ccctgatcgc
600 ttcacaggca gtggatctgg aacagatttc actctcacca tcagcagtgt
gcaggctgaa 660 gacctggcag tttattactg tcagaatgat tatagttatc
ctctcacgtt cggtgctggg 720 accaagcttg agatcaaacg tacgactagt
tccgggcatc atcaccatca tcat 774 71 258 PRT Artificial Sequence
Description of Artificial Sequence Synthetic P5-23 single chain Fv
71 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser
Arg Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Asp Ser Ser Thr Ile
Asn Tyr Thr Pro Ser Leu 50 55 60 Lys Asp Arg Phe Ile Ile Ser Arg
Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Lys Val
Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly
Gln Trp Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125
Gly Gly Gly Gly Ser Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu 130
135 140 Thr Val Thr Ala Gly Glu Arg Val Thr Met Ser Cys Lys Ser Ser
Gln 145 150 155 160 Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu
Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu
Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg
Phe Thr Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile
Ser Ser Val Gln Ala Glu Asp Leu Ala Val 210 215 220 Tyr Tyr Cys Gln
Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly 225 230 235 240 Thr
Lys Leu Glu Ile Lys Arg Thr Thr Ser Ser Gly His His His His 245 250
255 His His 72 1497 DNA Artificial Sequence Description of
Artificial Sequence Synthetic 3B10xP4-3 bispecific single chain Fv
72 gatattgtga tgacgcaggc tgcattctcc aatccagtca ctcttggaac
atcagcttcc 60 atctcctgca ggtctagtaa gagtctccta catagtaatg
gcatcactta tttgtattgg 120 tatctgcaga agccaggcca gtctcctcag
ctcctgattt atcagatgtc caaccttgcc 180 tcaggagtcc cagacaggtt
cagtagcagt gggtcaggaa ctgatttcac actgagaatc 240 agcagagtgg
aggctgagga tgtgggtgtt tattactgtg ctcaaaatct agaacttcct 300
cggacgttcg gtggaggcac caagctggaa atcaaaggtg gtggtggttc tggcggcggc
360 ggctccggtg gtggtggttc tcaggtgcaa ctgcagcagt cagggcctga
gctgaagaag 420 cctggagaga cagtcaagat ctcctgcaag gcttctgggt
ataccttcac aaactatgga 480 atgaactggg tgaagcaggc tccaggaaag
ggtttcaagt ggatgggctg gataaacacc 540 tacactggag agccaacata
tggtgatgac ttcaagggac ggtttgcctt ctctttggaa 600 acctctgcca
gcactgccta tttgcagatc aacaacctca aaaatgagga cacggctaca 660
tatttctgtg caagattcac ctcccctgac tactggggcc aagggaccac ggtcaccgtc
720 tcctccggag gtggtggatc cgaggtgcag ctgctcgagt ctggaggtgg
cctggtgcag 780 cctggaggat ccctgaaact ctcctgtgca gcctctggat
tcgattttag tagatactgg 840 atgagttggg tccggcaggc tccagggaaa
gggctagaat ggattggaga aattaatcca 900 gatagcagta cgataaacta
tacgccatct ctaaaggata aattcatcat ctccagagac 960 aacgccaaaa
atacgctgta cctgcaaatg agcaaagtga gatctgagga cacagccctt 1020
tattactgtg caagaggggc ggtagtagct ccctttgact actggggcca agggaccacg
1080 gtcaccgtct cctcaggtgg tggtggttct ggcggcggcg gctccggtgg
tggtggttct 1140 gagctcgtca tgacccagtc tccatcctcc ttatctgcct
ctctgggaga aagagtcagt 1200 ctcacttgtc gggcaagtca ggacattggt
agtagcttaa actggcttca gcaggaacca 1260 gatggaacta ttaaacgcct
gatctacgcc acatccagtt tagattctgg tgtccccaaa 1320 aggttcagtg
gcagtaggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 1380
gaagattttg tagactatta ctgtctacaa tatgctagtt ctccgtacac gttcggaggg
1440 gggaccaagc ttgagatcaa acgtacgact agttccgggc atcatcacca tcatcat
1497 73 499 PRT Artificial Sequence Description of Artificial
Sequence Synthetic 3B10xP4-3 bispecific single chain Fv 73 Asp Ile
Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly 1 5 10 15
Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20
25 30 Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser
Gly Val Pro 50 55 60 Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp
Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly
Val Tyr Tyr Cys Ala Gln Asn 85 90 95 Leu Glu Leu Pro Arg Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 115 120 125 Val Gln Leu
Gln Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr 130 135 140 Val
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly 145 150
155 160 Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Phe Lys Trp Met
Gly 165 170 175 Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Gly Asp
Asp Phe Lys 180 185 190 Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala
Ser Thr Ala Tyr Leu 195 200 205 Gln Ile Asn Asn Leu Lys Asn Glu Asp
Thr Ala Thr Tyr Phe Cys Ala 210 215 220 Arg Phe Thr Ser Pro Asp Tyr
Trp Gly Gln Gly Thr Thr Val Thr Val 225 230 235 240 Ser Ser Gly Gly
Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly 245 250 255 Gly Leu
Val Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser 260 265 270
Gly Phe Asp Phe Ser Arg Tyr Trp Met Ser Trp Val Arg Gln Ala Pro 275
280 285 Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Asn Pro Asp Ser Ser
Thr 290 295 300 Ile Asn Tyr Thr Pro Ser Leu Lys Asp Lys Phe Ile Ile
Ser Arg Asp 305 310 315 320 Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met
Ser Lys Val Arg Ser Glu 325 330 335 Asp Thr Ala Leu Tyr Tyr Cys Ala
Arg Gly Ala Val Val Ala Pro Phe 340 345 350 Asp Tyr Trp Gly Gln Gly
Thr Thr Val Thr Val Ser Ser Gly Gly Gly 355 360 365 Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val Met 370 375 380 Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly Glu Arg Val Ser 385 390 395
400 Leu Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Ser Leu Asn Trp Leu
405 410 415 Gln Gln Glu Pro Asp Gly Thr Ile Lys Arg Leu Ile Tyr Ala
Thr Ser 420 425 430 Ser Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly
Ser Arg Ser Gly 435 440 445 Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu
Glu Ser Glu Asp Phe Val 450 455 460 Asp Tyr Tyr Cys Leu Gln Tyr Ala
Ser Ser Pro Tyr Thr Phe Gly Gly 465 470 475 480 Gly Thr Lys Leu Glu
Ile Lys Arg Thr Thr Ser Ser Gly His His His 485 490 495 His His His
74 1509 DNA Artificial Sequence Description of Artificial Sequence
Synthetic 3B10xP4-14 bispecific single chain Fv 74 gatattgtga
tgacgcaggc tgcattctcc aatccagtca ctcttggaac atcagcttcc 60
atctcctgca ggtctagtaa gagtctccta catagtaatg gcatcactta tttgtattgg
120 tatctgcaga agccaggcca gtctcctcag ctcctgattt atcagatgtc
caaccttgcc 180 tcaggagtcc cagacaggtt cagtagcagt gggtcaggaa
ctgatttcac actgagaatc 240 agcagagtgg aggctgagga tgtgggtgtt
tattactgtg ctcaaaatct agaacttcct 300 cggacgttcg gtggaggcac
caagctggaa atcaaaggtg gtggtggttc tggcggcggc 360 ggctccggtg
gtggtggttc tcaggtgcaa ctgcagcagt cagggcctga gctgaagaag 420
cctggagaga cagtcaagat ctcctgcaag gcttctgggt ataccttcac aaactatgga
480 atgaactggg tgaagcaggc tccaggaaag ggtttcaagt ggatgggctg
gataaacacc 540 tacactggag agccaacata tggtgatgac ttcaagggac
ggtttgcctt ctctttggaa 600 acctctgcca gcactgccta tttgcagatc
aacaacctca aaaatgagga cacggctaca 660 tatttctgtg caagattcac
ctcccctgac tactggggcc aagggaccac ggtcaccgtc 720 tcctccggag
gtggtggatc cgaggtgcag ctgctcgagt ctggaggtgg cctggtgcag 780
cctggaggat ccctgaaact ctcctgtgca gcctcaggat tcgattttag tagatactgg
840 atgagttggg tccggcaggc tccagggaaa gggctagaat ggattggaga
aattaatcca 900 gatagcagta cgataaacta tacgccatct ctaaaggata
aattcatcat ctccagagac 960 aacgccaaaa atacgctgta cctgcaaatg
agcaaagtga gatctgagga cacagccctt 1020 tattactgtg caagacgcag
ctacggtagt agctacgact ggtacttcga tgtctggggc 1080 caagggacca
cggtcaccgt ctcctcaggt ggtggtggtt ctggcggcgg cggctccggt 1140
ggtggtggtt ctgagctcca gatgacccag tctccagcct ccctatctgc atctgtggga
1200 gaaactgtca ccatcacatg tcgagcaagt gagaatattt acagttattt
agcatggtat 1260 cagcagaaac agggaaaatc tcctcagctc ctggtctata
atgcaaaaac cttagcagaa 1320 ggtgtgccat caaggttcag tagcagtgga
tcaggcacac agttttctct gaagatcaac 1380 agcctgcagc ctgaagattt
tgggagttat tactgtcaac atcattatgg tactccgctc 1440 acgttcggtg
ctgggaccaa gcttgagatc aaacgtacga ctagttccgg gcatcatcac 1500
catcatcat 1509 75 503 PRT Artificial Sequence Description of
Artificial Sequence Synthetic 3B10xP4-14 bispecific single chain Fv
75 Asp Ile Val Met Thr Gln Ala Ala Phe Ser
Asn Pro Val Thr Leu Gly 1 5 10 15 Thr Ser Ala Ser Ile Ser Cys Arg
Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Ile Thr Tyr Leu
Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu
Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg
Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn 85
90 95 Leu Glu Leu Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gln 115 120 125 Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Lys
Lys Pro Gly Glu Thr 130 135 140 Val Lys Ile Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Asn Tyr Gly 145 150 155 160 Met Asn Trp Val Lys Gln
Ala Pro Gly Lys Gly Phe Lys Trp Met Gly 165 170 175 Trp Ile Asn Thr
Tyr Thr Gly Glu Pro Thr Tyr Gly Asp Asp Phe Lys 180 185 190 Gly Arg
Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu 195 200 205
Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Ala 210
215 220 Arg Phe Thr Ser Pro Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr
Val 225 230 235 240 Ser Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu
Glu Ser Gly Gly 245 250 255 Gly Leu Val Gln Pro Gly Gly Ser Leu Lys
Leu Ser Cys Ala Ala Ser 260 265 270 Gly Phe Asp Phe Ser Arg Tyr Trp
Met Ser Trp Val Arg Gln Ala Pro 275 280 285 Gly Lys Gly Leu Glu Trp
Ile Gly Glu Ile Asn Pro Asp Ser Ser Thr 290 295 300 Ile Asn Tyr Thr
Pro Ser Leu Lys Asp Lys Phe Ile Ile Ser Arg Asp 305 310 315 320 Asn
Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Lys Val Arg Ser Glu 325 330
335 Asp Thr Ala Leu Tyr Tyr Cys Ala Arg Arg Ser Tyr Gly Ser Ser Tyr
340 345 350 Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr
Val Ser 355 360 365 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser 370 375 380 Glu Leu Gln Met Thr Gln Ser Pro Ala Ser
Leu Ser Ala Ser Val Gly 385 390 395 400 Glu Thr Val Thr Ile Thr Cys
Arg Ala Ser Glu Asn Ile Tyr Ser Tyr 405 410 415 Leu Ala Trp Tyr Gln
Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 420 425 430 Tyr Asn Ala
Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Ser 435 440 445 Ser
Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro 450 455
460 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Leu
465 470 475 480 Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg Thr
Thr Ser Ser 485 490 495 Gly His His His His His His 500 76 1509 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
3B10xP5-2 bispecific single chain Fv 76 gatattgtga tgacgcaggc
tgcattctcc aatccagtca ctcttggaac atcagcttcc 60 atctcctgca
ggtctagtaa gagtctccta catagtaatg gcatcactta tttgtattgg 120
tatctgcaga agccaggcca gtctcctcag ctcctgattt atcagatgtc caaccttgcc
180 tcaggagtcc cagacaggtt cagtagcagt gggtcaggaa ctgatttcac
actgagaatc 240 agcagagtgg aggctgagga tgtgggtgtt tattactgtg
ctcaaaatct agaacttcct 300 cggacgttcg gtggaggcac caagctggaa
atcaaaggtg gtggtggttc tggcggcggc 360 ggctccggtg gtggtggttc
tcaggtgcaa ctgcagcagt cagggcctga gctgaagaag 420 cctggagaga
cagtcaagat ctcctgcaag gcttctgggt ataccttcac aaactatgga 480
atgaactggg tgaagcaggc tccaggaaag ggtttcaagt ggatgggctg gataaacacc
540 tacactggag agccaacata tggtgatgac ttcaagggac ggtttgcctt
ctctttggaa 600 acctctgcca gcactgccta tttgcagatc aacaacctca
aaaatgagga cacggctaca 660 tatttctgtg caagattcac ctcccctgac
tactggggcc aagggaccac ggtcaccgtc 720 tcctccggag gtggtggatc
cgaggtgcag ctgctcgagg agtctggagg aggcttggtg 780 caacctggag
gatccatgaa actctcctgt gttgcctctg gattcacttt cagtaactac 840
tggatgaact gggtccgcca gtctccagag aaggggcttg agtgggttgc tgaaattaga
900 ttgaaatcta ataattatgc aacacattat gcggagtctg tgaaagggag
gttcaccatc 960 tcaagagatg attccaaaag tagtgtctac ctgcaaatga
acaacttaag agctgaagac 1020 actggcattt attactgtac caggctcccc
tacggctttg ctatggacta ctggggccaa 1080 gggaccacgg tcaccgtctc
ctcaggtggt ggtggttctg gcggcggcgg ctccggtggt 1140 ggtggttctg
agctcgtgct cacccagtct ccaaccacca tggctgcatc tcccggggag 1200
aagatcacta tcacctgcag tgccagctca agtataagtt ccaattactt gcattggtat
1260 cagcagaagc caggattctc ccctaaactc ttgatttata ggacatccaa
tctggcttct 1320 ggagtcccag ctcgcttcag tggcagtggg tctgggacct
cttactctct cacaattggc 1380 accatggagg ctgaagatgt tgccacttac
tactgccagc agggtagtag tataccgctc 1440 acgttcggtg ctgggaccaa
gcttgagatc aaacgtacga ctagttccgg gcatcatcac 1500 catcatcat 1509 77
503 PRT Artificial Sequence Description of Artificial Sequence
Synthetic 3B10xP5-2 bispecific single chain Fv 77 Asp Ile Val Met
Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly 1 5 10 15 Thr Ser
Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30
Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr
Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Ala Gln Asn 85 90 95 Leu Glu Leu Pro Arg Thr Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gln 115 120 125 Val Gln Leu Gln Gln
Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr 130 135 140 Val Lys Ile
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly 145 150 155 160
Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Phe Lys Trp Met Gly 165
170 175 Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Gly Asp Asp Phe
Lys 180 185 190 Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr
Ala Tyr Leu 195 200 205 Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala
Thr Tyr Phe Cys Ala 210 215 220 Arg Phe Thr Ser Pro Asp Tyr Trp Gly
Gln Gly Thr Thr Val Thr Val 225 230 235 240 Ser Ser Gly Gly Gly Gly
Ser Glu Val Gln Leu Leu Glu Glu Ser Gly 245 250 255 Gly Gly Leu Val
Gln Pro Gly Gly Ser Met Lys Leu Ser Cys Val Ala 260 265 270 Ser Gly
Phe Thr Phe Ser Asn Tyr Trp Met Asn Trp Val Arg Gln Ser 275 280 285
Pro Glu Lys Gly Leu Glu Trp Val Ala Glu Ile Arg Leu Lys Ser Asn 290
295 300 Asn Tyr Ala Thr His Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr
Ile 305 310 315 320 Ser Arg Asp Asp Ser Lys Ser Ser Val Tyr Leu Gln
Met Asn Asn Leu 325 330 335 Arg Ala Glu Asp Thr Gly Ile Tyr Tyr Cys
Thr Arg Leu Pro Tyr Gly 340 345 350 Phe Ala Met Asp Tyr Trp Gly Gln
Gly Thr Thr Val Thr Val Ser Ser 355 360 365 Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 370 375 380 Leu Val Leu Thr
Gln Ser Pro Thr Thr Met Ala Ala Ser Pro Gly Glu 385 390 395 400 Lys
Ile Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Ser Ser Asn Tyr 405 410
415 Leu His Trp Tyr Gln Gln Lys Pro Gly Phe Ser Pro Lys Leu Leu Ile
420 425 430 Tyr Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe
Ser Gly 435 440 445 Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Gly
Thr Met Glu Ala 450 455 460 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln
Gly Ser Ser Ile Pro Leu 465 470 475 480 Thr Phe Gly Ala Gly Thr Lys
Leu Glu Ile Lys Arg Thr Thr Ser Ser 485 490 495 Gly His His His His
His His 500 78 1515 DNA Artificial Sequence Description of
Artificial Sequence Synthetic 3B10xP5-23 bispecific single chain Fv
78 gatattgtga tgacgcaggc tgcattctcc aatccagtca ctcttggaac
atcagcttcc 60 atctcctgca ggtctagtaa gagtctccta catagtaatg
gcatcactta tttgtattgg 120 tatctgcaga agccaggcca gtctcctcag
ctcctgattt atcagatgtc caaccttgcc 180 tcaggagtcc cagacaggtt
cagtagcagt gggtcaggaa ctgatttcac actgagaatc 240 agcagagtgg
aggctgagga tgtgggtgtt tattactgtg ctcaaaatct agaacttcct 300
cggacgttcg gtggaggcac caagctggaa atcaaaggtg gtggtggttc tggcggcggc
360 ggctccggtg gtggtggttc tcaggtgcaa ctgcagcagt cagggcctga
gctgaagaag 420 cctggagaga cagtcaagat ctcctgcaag gcttctgggt
ataccttcac aaactatgga 480 atgaactggg tgaagcaggc tccaggaaag
ggtttcaagt ggatgggctg gataaacacc 540 tacactggag agccaacata
tggtgatgac ttcaagggac ggtttgcctt ctctttggaa 600 acctctgcca
gcactgccta tttgcagatc aacaacctca aaaatgagga cacggctaca 660
tatttctgtg caagattcac ctcccctgac tactggggcc aagggaccac ggtcaccgtc
720 tcctccggag gtggtggatc cgaggtgcag ctgctcgagt ctggaggtgg
cctggtgcag 780 cctggaggat ccctgaaact ctcctgtgca gcctcaggat
tcgattttag tagatactgg 840 atgagttggg tccggcaggc tccagggaaa
gggctagaat ggattggaga aattaatcca 900 gatagcagta cgataaacta
tacgccatct ctaaaggata gattcatcat ctccagagac 960 aacgccaaaa
atacgctgta cctgcaaatg agcaaagtga ggtctgagga cacagccctt 1020
tattactgtg caagattggg gcaatggggg tactttgact actggggcca agggaccacg
1080 gtcaccgtct cctcaggtgg tggtggttct ggcggcggcg gctccggtgg
tggtggttct 1140 gagctcgtga tgacacagtc tccatcctcc ctgactgtga
cagcaggaga gagggtcact 1200 atgagctgca agtccagtca gagtctgtta
aacagtggaa atcaaaagaa ctacttgacc 1260 tggtaccagc agaaaccagg
gcagcctcct aaactgttga tctactgggc atccactagg 1320 gaatctgggg
tccctgatcg cttcacaggc agtggatctg gaacagattt cactctcacc 1380
atcagcagtg tgcaggctga agacctggca gtttattact gtcagaatga ttatagttat
1440 cctctcacgt tcggtgctgg gaccaagctt gagatcaaac gtacgactag
ttccgggcat 1500 catcaccatc atcat 1515 79 505 PRT Artificial
Sequence Description of Artificial Sequence Synthetic 3B10xP5-23
bispecific single chain Fv 79 Asp Ile Val Met Thr Gln Ala Ala Phe
Ser Asn Pro Val Thr Leu Gly 1 5 10 15 Thr Ser Ala Ser Ile Ser Cys
Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Asn Gly Ile Thr Tyr
Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu
Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp
Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70
75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln
Asn 85 90 95 Leu Glu Leu Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gln 115 120 125 Val Gln Leu Gln Gln Ser Gly Pro Glu
Leu Lys Lys Pro Gly Glu Thr 130 135 140 Val Lys Ile Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Asn Tyr Gly 145 150 155 160 Met Asn Trp Val
Lys Gln Ala Pro Gly Lys Gly Phe Lys Trp Met Gly 165 170 175 Trp Ile
Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Gly Asp Asp Phe Lys 180 185 190
Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu 195
200 205 Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys
Ala 210 215 220 Arg Phe Thr Ser Pro Asp Tyr Trp Gly Gln Gly Thr Thr
Val Thr Val 225 230 235 240 Ser Ser Gly Gly Gly Gly Ser Glu Val Gln
Leu Leu Glu Ser Gly Gly 245 250 255 Gly Leu Val Gln Pro Gly Gly Ser
Leu Lys Leu Ser Cys Ala Ala Ser 260 265 270 Gly Phe Asp Phe Ser Arg
Tyr Trp Met Ser Trp Val Arg Gln Ala Pro 275 280 285 Gly Lys Gly Leu
Glu Trp Ile Gly Glu Ile Asn Pro Asp Ser Ser Thr 290 295 300 Ile Asn
Tyr Thr Pro Ser Leu Lys Asp Arg Phe Ile Ile Ser Arg Asp 305 310 315
320 Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Lys Val Arg Ser Glu
325 330 335 Asp Thr Ala Leu Tyr Tyr Cys Ala Arg Leu Gly Gln Trp Gly
Tyr Phe 340 345 350 Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser
Ser Gly Gly Gly 355 360 365 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Glu Leu Val Met 370 375 380 Thr Gln Ser Pro Ser Ser Leu Thr
Val Thr Ala Gly Glu Arg Val Thr 385 390 395 400 Met Ser Cys Lys Ser
Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys 405 410 415 Asn Tyr Leu
Thr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu 420 425 430 Leu
Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe 435 440
445 Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val
450 455 460 Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr
Ser Tyr 465 470 475 480 Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu
Ile Lys Arg Thr Thr 485 490 495 Ser Ser Gly His His His His His His
500 505 80 32 DNA Artificial Sequence Description of Artificial
Sequence Synthetic oligonucleotide 80 atcaagcttg tggatatgtt
acaaaaataa ct 32 81 30 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 81 atcaagcttg
aaccaagaag ttcaaattcc 30 82 34 DNA Artificial Sequence Description
of Artificial Sequence Synthetic oligonucleotide 82 cgcggtggcg
gccgcttaca cagtcctttg catg 34 83 37 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 83
aggtgtacac tccgatatcc agctgaccca gtctcca 37 84 51 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 84 ggagccgccg ccgccagaac caccaccacc tttgatctcg
agcttggtcc c 51 85 53 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 85 ggcggcggcg
gctccggtgg tggtggttct caggtsmarc tgcagsagtc wgg 53 86 39 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 86 aatccggagg agacggtgac cgtggtccct tggccccag 39 87
39 DNA Artificial Sequence Description of Artificial Sequence
Primer 87 aggtgtacac tccttattca accaagaagt tcaaattcc 39 88 31 DNA
Artificial Sequence Description of Artificial Sequence Primer 88
tcatccggac acagtccttt gcatgcagat g 31 89 504 DNA Artificial
Sequence Description of Artificial Sequence Synthetic soluble NKG2D
nucleotide sequence 89 gaattcacc atg gga tgg agc tgt atc atc ctc
ttc ttg gta gca aca gct 51 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu
Val Ala Thr Ala 1 5 10 aca ggt gta cac tcc tta ttc aac caa gaa gtt
caa att ccc ttg acc 99 Thr Gly Val His Ser Leu Phe Asn Gln Glu Val
Gln Ile Pro Leu Thr 15 20 25 30 gaa agt tac tgt ggc cca tgt cct aaa
aac tgg ata tgt tac aaa aat 147 Glu Ser Tyr Cys Gly Pro Cys Pro Lys
Asn Trp Ile Cys Tyr Lys Asn 35 40 45 aac tgc tac caa ttt ttt gat
gag agt aaa aac tgg tat gag agc cag 195 Asn Cys Tyr Gln Phe Phe Asp
Glu Ser Lys Asn Trp Tyr Glu Ser Gln 50 55 60 gct tct tgt atg tct
caa aat gcc agc ctt ctg aaa gta tac agc aaa 243 Ala Ser Cys Met Ser
Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys 65 70 75 gag gac cag
gat tta ctt aaa ctg gtg aag tca tat cat tgg atg gga 291
Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly 80
85 90 cta gta cac att cca aca aat gga tct tgg cag tgg gaa gat ggc
tcc 339 Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly
Ser 95 100 105 110 att ctc tca ccc aac cta cta aca ata att gaa atg
cag aag gga gac 387 Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu Met
Gln Lys Gly Asp 115 120 125 tgt gca ctc tat gcc tcg agc ttt aaa ggc
tat ata gaa aac tgt tca 435 Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly
Tyr Ile Glu Asn Cys Ser 130 135 140 act cca aat aca tac atc tgc atg
caa agg act gtg tcc ggg cat cat 483 Thr Pro Asn Thr Tyr Ile Cys Met
Gln Arg Thr Val Ser Gly His His 145 150 155 cac cat cat cat
tgagtcgac 504 His His His His 160 90 162 PRT Artificial Sequence
Description of Artificial Sequence Synthetic soluble NKG2D amino
acid sequence 90 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala
Thr Ala Thr Gly 1 5 10 15 Val His Ser Leu Phe Asn Gln Glu Val Gln
Ile Pro Leu Thr Glu Ser 20 25 30 Tyr Cys Gly Pro Cys Pro Lys Asn
Trp Ile Cys Tyr Lys Asn Asn Cys 35 40 45 Tyr Gln Phe Phe Asp Glu
Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser 50 55 60 Cys Met Ser Gln
Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp 65 70 75 80 Gln Asp
Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val 85 90 95
His Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu 100
105 110 Ser Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys
Ala 115 120 125 Leu Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys
Ser Thr Pro 130 135 140 Asn Thr Tyr Ile Cys Met Gln Arg Thr Val Ser
Gly His His His His 145 150 155 160 His His 91 5 PRT Artificial
Sequence Description of Artificial Sequence Synthetic Gly-Ser
linker 91 Gly Gly Gly Gly Ser 1 5 92 15 PRT Artificial Sequence
Description of Artificial Sequence Synthetic Gly-Ser linker 92 Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10
15
* * * * *
References